EP3313439A2 - Vaccins contre la grippe à correspondance antigénique - Google Patents

Vaccins contre la grippe à correspondance antigénique

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Publication number
EP3313439A2
EP3313439A2 EP16738554.1A EP16738554A EP3313439A2 EP 3313439 A2 EP3313439 A2 EP 3313439A2 EP 16738554 A EP16738554 A EP 16738554A EP 3313439 A2 EP3313439 A2 EP 3313439A2
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EP
European Patent Office
Prior art keywords
vaccine
influenza
antigen
virus
viruses
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Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP16738554.1A
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German (de)
English (en)
Inventor
Ethan C. SETTEMBRE
Philip R. Dormitzer
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Seqirus UK Ltd
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Seqirus UK Ltd
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Publication of EP3313439A2 publication Critical patent/EP3313439A2/fr
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/145—Orthomyxoviridae, e.g. influenza virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5252—Virus inactivated (killed)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55566—Emulsions, e.g. Freund's adjuvant, MF59
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/70—Multivalent vaccine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16121—Viruses as such, e.g. new isolates, mutants or their genomic sequences
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • This invention relates generally to vaccines, more specifically to improved influenza vaccines.
  • a major challenge for influenza vaccine manufacturers is that influenza viruses are constantly changing, thus requiring influenza vaccines to be adapted to the circulating strains each year.
  • WHO World Health Organization
  • influenza strains chosen by the WHO have generally proven to be a good match for the circulating influenza strains, there have been occasions where the circulating strains no longer matched the influenza strain found in the vaccine well and where the influenza vaccine therefore had a lower efficacy. For example, in the 2014/2015 season there was a mismatch between the H3N2 strain selected for the vaccine and the circulating H3N2 strain, resulting in the vaccine providing poor protection against this strain.
  • the present invention is based at least in part on the recognition that seasonal influenza vaccines in certain influenza seasons lack desirable vaccine efficacy (in clinical context) or vaccine effectiveness (in epidemiological context) due to antigenic mismatch between vaccine strains and circulating viruses. In recent years, ineffective flu vaccines have been observed in an alarming number of flu seasons.
  • the present invention provides solutions to the existing unmet needs for improved influenza vaccines with better antigenic match, for example, in the form of rescue vaccines and hybrid vaccines, which are described in more detail herein.
  • the rescue vaccine and the hybrid vaccine have in common that both are contemplated to include an antigen that has been produced in an egg-free process, and both include an antigen that provides antigenic match to circulating viruses in the way conventional egg-based vaccines cannot achieve.
  • Rescue vaccines are typically produced off-cycle, following a regular seasonal flu vaccine that has been found to be ineffective in providing sufficient immuno-protection due to antigenic mismatch.
  • a rescue vaccine may be used as a "follow-up" vaccine to supplement regular seasonal vaccines that are already commercially available, or already being manufactured, earlier in the same flu season.
  • a rescue vaccine may be a monovalent, cell culture-based vaccine.
  • Such vaccines may be made available weeks after regular seasonal vaccines are made available, e.g., 4 weeks, or, e.g. 8 weeks, 10 weeks, 12 weeks, 16 weeks, 20 weeks, 24 weeks.
  • rescue vaccines may provide a solution to antigenic mismatch stemming from egg adaptation.
  • Rescue vaccines described herein may also, or alternatively, provide a solution to antigenic mismatch stemming from antigenic drift or shift of circulating viruses (e.g. between selection of strains for vaccine production and the actual peak of disease).
  • a first influenza vaccine as described herein may be a vaccine comprising an antigen from a first influenza virus which has been passaged in eggs.
  • a first influenza vaccine may be made available prior to a second influenza vaccine.
  • a rescue vaccine as described herein may be a second influenza vaccine rescue vaccine as described herein.
  • the second (rescue) vaccine may comprise an antigen from a second influenza virus which has not been passaged in eggs, wherein the antigen in the second influenza vaccine is more closely antigenically matched to a circulating strain than the antigen in the first influenza vaccine.
  • the second influenza vaccine may be administered within 1 month, 2 months, 3 months, 4 months or 5 months after the first influenza vaccine.
  • the second influenza vaccine is administered within 3 months after the first influenza vaccine.
  • a rescue influenza vaccine comprises an antigen prepared in host cells, wherein the antigen has greater (i.e. closer) antigenic match to antigens of circulating influenza viruses (e.g. an antigen of a circulating strain) than a seasonal influenza vaccine (e.g. a first influenza vaccine) available earlier in the same influenza season, wherein the seasonal influenza vaccine has ⁇ 50% vaccine effectiveness against the circulating influenza virus.
  • circulating influenza viruses e.g. an antigen of a circulating strain
  • a seasonal influenza vaccine e.g. a first influenza vaccine
  • the invention further provides hybrid vaccines and its intermediate compositions (for example, intermediate compositions prepared during manufacture of the hybrid vaccine, prior to the final vaccine product).
  • a hybrid vaccine can comprise at least one antigen that is produced in a non-egg- based preparation and at least one antigen that is produced in an egg-based preparation.
  • Conventional egg-based production may be employed to manufacture antigens of strains that are not susceptible to egg adaptation or clade mismatch.
  • one or more antigens of strains that are susceptible to egg-specific challenges are produced in egg-free system(s).
  • a hybrid vaccine may combine such components into preferably a single formulation so as to circumvent egg-dependent antigenic mismatch.
  • a hybrid vaccine comprises two or more components selected from the group consisting of: (i) a viral antigen from an egg-based preparation; (ii) a viral antigen from a cell culture-based preparation; (iii) a viral antigen from a recombinant protein preparation; and (iv) an RNA replicon encoding a viral antigen.
  • the invention provides improved seed virus and related methods, for producing antigenically matched vaccines. This is based at least in part on the recognition that the presence of mixed species (e.g., quasi-species) in original isolates is likely a contributing factor to subsequent antigenic drifting. This is contrary to what has been widely considered a consequence of so-called "cell adaptation.” Accordingly, the invention provides solutions to counter the source of the problem by using synthetic viruses made with defined genetic sequences. Thus, the invention provides improved seed virus, candidate vaccine virus (CVV) or reference strains, which are less susceptible to antigenic drifting than wild-type isolates.
  • CVV candidate vaccine virus
  • a seed virus preparation described herein comprises a genetically homogeneous population of viruses, wherein an antigen of the population of viruses is synthetically derived from a genetically defined sequence.
  • the invention also encompasses use of the seed virus preparation described herein in the manufacture of a composition.
  • the seed virus preparation may be used to prepare or manufacture a composition, an influenza vaccine, a rescue vaccine or a hybrid vaccine, as defined herein.
  • the invention provides a vaccine made by a process comprising a step of making a seed virus preparation comprising a genetically homogeneous population of viruses, characterized in that an antigen of the population of viruses is synthetically derived from a genetically defined sequence.
  • the invention also provides improved influenza vaccines and related uses and manufacturing processes for the same.
  • Figure 1 provides a line graph illustrating that worst flu seasons in recent years are those where virus has evolved (i.e. those in which the virus has undergone the greatest change). Time course of hospital visits associated with flu-like illness for each of the six flu seasons is plotted over time.
  • Figure 2 provides a graph showing the relationship between vaccine efficacy and influenza-related hospitalization rate for different age groups. (Adapted from: Nichol K, et al. Vaccine 2003; 21 :1769-1775; Goodwin K, et al. Vaccine 2006; 24:1 159-1 169; Grubeck-Loebenstein B, et al. Nat Med 1998; 4:870; and, Glezen WP, et al. Am Rev Respir Dis 1987; 136:550-555.)
  • Figure 3 provides a bar graph depicting H3N2 viral isolation rates in four different systems.
  • Figure 4 provides a graph showing HA titers (in the presence of oseltamivir) of 3C.2A viruses that were serially passaged in MDCK cells.
  • Figure 5 provides a panel of synthetic viruses generated for antigenic characterization.
  • "Egg” or “cell” under antigen source refers to the passage history of the viruses that provided the HA and NA sequences for synthesis. In cases of mixed passage history, any passage in eggs is sufficient to trigger an "egg” designation. All synthetic test viruses were passaged exclusively in mammalian cells for these studies, regardless of the HA and NA sequences used.
  • Figure 6 provides HI comparison of egg-propagated non-synthetic viruses and MDCK cell- propagated synthetic viruses with HA and NA sequences derived from egg-adapted candidate vaccine viruses.
  • Figure 7 provides HI characterization of synthetic viruses with HA and NA sequences derived from egg- or mammalian-propagated wild-type viruses.
  • Figure 8 provides HI titers showing antigenic mismatch between viruses with egg- and mammalian cell-derived H3N2 antigens: (A) A/Victoria/210/2009 (H3N2); and, (B) A/Victoria/361/201 1 (H3N2).
  • Figure 9 provides HI titers showing antigenic mismatch between viruses with egg- and mammalian cell-derived B antigens.
  • Figure 10 provides Two-way HI test for the H3N2 strain A/Switzerland/9715293/2013.
  • Figure 11 provides micro-neutralization assay results for mouse antisera raised against egg- and cell-derived H3N2 monobulks derived from three major clades (3C.1 , 3C.2a, 3C.3a), with or without adjuvant, and tested against: (A) egg-derived A/Hong Kong/5738/2014 test virus; and (B) cell-derived A/Hong Kong/5738/2014 test virus.
  • 3C.1 , 3C.2a, 3C.3a three major clades
  • the present invention is based at least in part on the recognition that seasonal influenza vaccines in certain influenza seasons lack desirable efficacy due to antigenic mismatch between a vaccine antigen and circulating strains.
  • the terms "match” and “mismatch,” unless otherwise specified, refer to antigenic match and mismatch, respectively, between corresponding antigens of the vaccine strain (e.g., “candidate vaccine virus” or “CVV”) and the circulating strain.
  • Antigenic mismatch can result in low effectiveness of the vaccine in providing protection for vaccinated individuals. In certain seasons, for example, the efficacy may be 50% or less, e.g., between 0-50% due to antigenic mismatch (see more in Examples below). Low effectiveness may be defined as ⁇ 50% vaccine effectiveness (or even ⁇ 30% vaccine effectiveness) against the circulating influenza virus.
  • antigenic relatedness or “antigenic similarity”, which are used interchangeably to refer to degree of “antigenic match”.
  • Hemagglutinin is a primary antigen of influenza virus.
  • the head region of the HA protein is the dominant antigenic region and contains key epitopes, as well as receptor binding site required for host cell entry.
  • HI hemagglutination inhibition
  • Table 1 provides a complete "Two way” table showing the relatedness of the following viruses: WI/05 (A/Wisconsin/67/2005); NY/04 (A/New York/55/2004); WY/03 (A/Wyoming/3/2003); and, PM/99 (A/Panama/2007/1999).
  • Table 1 Exemplary two-way HI test.
  • viruses with ⁇ 4 fold differences in HI are considered “similar” (i.e. antigenically similar) (e.g., WY/03 and PM/99).
  • the HI assay essentially measures (i) existence, (ii) accessibility, and (iii) immunodominance of epitopes that HA contribute to antigenicity.
  • HI is a functional assay to determine if an antigen is likely raise an immune response and provides information on antigenic relatedness (e.g., match or mismatch).
  • Micro-neutralization (“MN”) assay is an alternative means for characterizing the antigenicity of viruses.
  • the MN assays may be used in place of HI assays, particularly for certain viruses that do not have HA with the ability to sufficiently bind to red blood cells, since HI assays are useful only if the HA of the virus is capable of agglutinating red blood cells.
  • certain H3N2 viruses such as 3C.2A viruses do not bind red blood cells with their HA.
  • antigenic match of antigen from such viruses is determined by MN assay.
  • the MN assay is a highly sensitive and specific assay for detecting virus-specific neutralizing antibodies to influenza viruses in human or animal sera, (ref: WHO Global Influenza Surveillance Network: Manual for the laboratory diagnosis and virological surveillance of influenza; 201 1 ; ISBN 978 92 4 154809 0; available at http://apps.who.int/iris/bitstream/10665/44518/1/9789241548090_eng.pdf).
  • the MN assay is based on the assumption that serum-neutralizing antibodies to influenza viral HA will inhibit the infection of MDCK cells with virus. Serially diluted sera are pre-incubated with a standardized amount of virus before the addition of MDCK cells.
  • the cells After overnight incubation, the cells are fixed, and the presence of virus- infected cells is detected by an anti-influenza nucleoprotein ELISA or by plaque reduction.
  • the absence of infectivity constitutes a positive neutralization reaction and indicates the presence of specific antibodies in the serum sample that can bind to the virus.
  • the neutralization titer is expressed as the reciprocal of the highest dilution of sera at which virus infection is blocked. If a test serum produces a neutralization titer that is similar for two viruses, it means that the antibodies in the test sera can effectively cross-react with both viruses, suggesting that the two viruses are antigenically- related.
  • a person skilled in the art may use standard assays to assess whether one influenza antigen or vaccine is more closely antigenically matched to a reference strain (e.g. a circulating strain) than another antigen or vaccine.
  • Antigenic match may be determined using a HI assay.
  • antigenic match may be determined using a MN assay.
  • the antigen in the second influenza vaccine is more closely antigenically matched to the circulating strain than the antigen in the first influenza vaccine if antibodies raised against the antigen in the second influenza vaccine show a higher degree of cross-reaction in the assay compared to antibodies raised against the antigen in the first influenza vaccine under identical conditions.
  • antigenic mismatch may result from incorrect prediction as to what viruses are most likely to cause illness in the coming season.
  • Viruses in the seasonal flu vaccine are selected each year based on surveillance-based forecasts about viruses, and WHO recommends specific vaccine viruses for inclusion in influenza vaccines, but then each individual country makes their own decision for which strains should be included in influenza vaccines licensed in their country.
  • a "recommended" strain (or a "reference” strain) based on which regular seasonal influenza vaccines are produced by vaccine manufacturers, may differ from predominant strains in circulation in the flu season. In some cases, however, influenza vaccines that are manufactured in accordance with correct recommendations from WHO may still result in vaccines with low efficacy. This is in part due to a phenomenon referred to as egg adaptation.
  • Egg adaptation may occur due to egg-specific genetic selection when a virus is propagated or grown in eggs. The virus must then adapt to the host cellular environment and genetic background, resulting in antigenic mismatch between the viral antigen prepared in the egg-based system and the corresponding antigen of circulating or recommended viruses.
  • a recommended strain may not grow well in an egg-based system. Indeed, certain clades of influenza viruses are known to grow poorly in eggs. In these situations, a different but related virus, for example a virus from a different but related clade, may be selected and recommended for the manufacture of flu vaccines in order to accommodate the egg- based production of vaccines. This results in clade mismatch, which may cause antigenic mismatch.
  • the present disclosure provides non-egg-based vaccines that enable improved antigenic match to achieve immunoprotection that egg-based vaccines may fail to provide.
  • influenza vaccine system has been developed to respond to the natural evolution of influenza viruses, but the problem of antigenic mismatch continues to be a challenge in certain years.
  • mismatches arise naturally due to the antigenic drift of circulating viruses after vaccine strain selection has already been made.
  • mismatches are introduced as part of the current system, which relies on the use of egg-adapted isolates as a starting material for candidate vaccine viruses. Recognition of the source of the existing problems and transforming the current process for making vaccine viruses may be of great value towards improving public health.
  • the inventors of the present invention have tested the idea that this technology may also be used to produce viruses that maintain antigenic match to the intended reference viruses (e.g., matching to a circulating strain), depending on the hemagglutinin (HA) and neuraminidase (NA) sequences used for gene synthesis.
  • HA hemagglutinin
  • NA neuraminidase
  • Work disclosed herein demonstrates general utility of this approach. Briefly, a panel of synthetic viruses was generated, using HA and NA sequences from recent isolates. As evaluated by hemagglutination inhibition tests, synthetic viruses are shown to be antigenically similar to the conventional egg- or cell-propagated reference strains.
  • wild-type 3C.2A viruses maintained in cell culture show HA and/or NA mutations, which can change receptor binding properties on host cells or red blood cells (RBCs).
  • RBCs red blood cells
  • the invention encompasses improved candidate vaccine viruses and related methods of producing candidate vaccine viruses using synthetic seed virus.
  • Synthetic seed virus technology may be used to obtain a genetically homogenous preparation of seed virus, which can be used to produce a vaccine (e.g. a vaccine as defined herein).
  • the invention also includes improved seed viruses passaged in non-egg-based host system(s), such as a cell culture. Suitable cell cultures include eukaryotic cells, such as mammalian cells, insect cells, avian cells, etc. (as discussed further herein).
  • Such synthetic seed viruses are characterized in that they are genetically stable and typically grow to higher titers than viruses passaged in eggs. Genetic stability as used herein refers to resistance to mutations/adaptation after several rounds of passaging in cell culture, e.g., 2, 3, 4, 5, or 6 passages.
  • each preparation consists of a genetically homogeneous population of viruses (as opposed to mixed (i.e., heterogeneous) sub-populations, which may be the case in conventional seed virus preparations).
  • synthetic seed virus preparations may be determined to be genetically homogeneous based on DNA Sanger Sequencing (but not necessarily Next Generation Deep Sequencing).
  • a genetically homogeneous synthetic seed virus preparation may not be 100% genetically 'pure'. Indeed, a single round of passaging in cells may lead to a small amount of sequence diversification.
  • a genetically homogeneous synthetic seed virus preparation may contain a small proportion (e.g. less than 10%, more preferably less than 5%, 3% or 1 %) of synthetic virus that is not genetically identical to the predominant synthetic virus in the population.
  • the invention includes a method for manufacturing a vaccine (which may be an influenza vaccine for use according to the present invention), comprising a step of preparing a synthetic seed virus in an egg-free process, wherein the synthetic seed virus is not passaged in eggs.
  • an antigen prepared from the synthetic seed virus (which may be an antigen of a second influenza vaccine, as defined herein) is more closely antigenically matched to a reference strain (e.g. a circulating strain), as compared to an antigen from an egg-based counterpart (e.g. a first influenza vaccine, as defined herein).
  • the synthetic seed virus is isolated from a strain that (i) is susceptible to egg-adaptation; and/or (ii) has an HA that does not bind well to red blood cells.
  • the expression "does not bind well to red blood cells” means that the HA of that particular viral strain/clade has poor affinity for red blood cells such that it renders the standard HI assay unreliable.
  • the vaccine comprises an antigen from H3N2 and/or B strain(s).
  • the antigen is HA, NA, or combination thereof.
  • the invention thus provides a seed virus preparation comprising a genetically homogenous population of viruses, characterized in that HA of the genetically homogeneous population of viruses is synthetically prepared from a genetically defined sequence.
  • the invention also encompasses use of the seed virus preparation in the manufacture of a composition.
  • the invention provides a vaccine made by a process comprising a step of making a seed virus preparation comprising a genetically homogenous population of viruses, characterized in that an antigen of the genetically homogenous population of viruses is synthetically prepared from a genetically defined sequence.
  • the antigen is HA, NA, or combination thereof.
  • the vaccine is manufactured in an egg-free process, such that the virus is never passaged in eggs through the entire vaccine manufacture process.
  • the genetically defined sequence may, for example, be a HA sequence of a cell-based isolate, which may be an isolate as described herein.
  • synthetic seed virus as described above, may be used for the manufacture of a composition, a second influenza virus vaccine, a rescue vaccine or a hybrid vaccine, as defined herein.
  • the synthetic seed virus for use in the invention may be manufactured in an egg-free process, and may not have been passaged in eggs.
  • the synthetic seed virus is grown in cell culture.
  • the synthetic seed virus may be generated using HA and NA sequences from an isolate of a reference influenza virus strain (e.g. a circulating virus strain).
  • a reference influenza virus strain e.g. a circulating virus strain
  • the isolate is not egg-adapted.
  • the isolate is preferably isolated in cell culture (e.g. in mammalian cell lines qualified for vaccine production).
  • the isolate may not be a high-growth egg-based reassortant.
  • the isolate is preferably an isolate of a circulating strain.
  • the isolate may be included in a list of the predominant circulating influenza strains for a given influenza season (e.g. a list published by the WHO and/or the FDA).
  • the isolate may be isolated from clinical samples by one or more World Health Organization (WHO) National Influenza Centers.
  • WHO World Health Organization
  • the isolate may be an isolate of a clade or strain that is susceptible to egg adaptation and/or to clade mismatch.
  • a synthetic seed virus is typically prepared using reverse genetics techniques as described herein.
  • the virus may be prepared using a viral backbone comprising a set of viral genome segments encoding influenza virus proteins (other than HA and NA).
  • a synthetic seed virus is an A strain virus (e.g. H3N2 virus)
  • the synthetic seed virus may be prepared using a backbone selected from PR8, PR8x, #19, and #21 .
  • the strain is an H3N2 strain and the backbone is PR8x.
  • the synthetic seed virus is a B strain virus (e.g. a strain from the B/Victoria lineage)
  • the synthetic seed virus may be prepared using a backbone including all six backbone segments from B/Brisbane/60/2008.
  • the present invention provides such vaccines referred to herein as the rescue vaccine.
  • a rescue vaccine may be manufactured as a corrective measure to supplement a regular seasonal vaccine that has been shown to lack desired effectiveness, e.g., within the same flu season.
  • regular seasonal vaccines may be produced conventionally in accordance with WHO recommendation and are commercially made available for vaccination.
  • VE vaccine effectiveness
  • a rescue vaccine may be produced as a follow-up measure within the same flu season in non-egg-based preparation, which would provide better antigenic match to circulating viruses. Therefore, it is envisaged that rescue vaccines are prepared or propagated without eggs.
  • a rescue vaccine comprises (a) an antigen produced in a cell culture-based preparation, such as avian cell cultures and mammalian cell cultures; (b) a recombinant protein-based antigen; (c) an RNA replicon (e.g., self-amplifying RNA) encoding an antigen.
  • a rescue vaccine comprises any combinations thereof.
  • the rescue vaccine may comprise one, more than one, or all of (a), (b) and (c).
  • the recombinant protein-based antigen may be a recombinantly expressed antigen.
  • the antigen is an influenza antigen.
  • the invention provides a rescue influenza vaccine comprising an antigen prepared in host cells, wherein the antigen has greater (i.e. closer) antigenic match to antigens of circulating influenza viruses (e.g. an antigen of a circulating strain) than a seasonal influenza vaccine available earlier in the same influenza season, wherein the seasonal influenza vaccine has ⁇ 50% vaccine effectiveness against the circulating influenza viruses.
  • the antigen may be an antigen, as described herein, which has not been passaged in eggs.
  • the seasonal influenza vaccine may be a first influenza vaccine as described herein.
  • synthetic seed virus may be used to produce a rescue vaccine of the present invention.
  • the synthetic seed virus may be a synthetic seed virus provided herein.
  • the preparation of synthetic seeds can be faster, allowing temporal adjustments.
  • the use of the synthetic seed virus technology enables a genetically homogenous preparation of seed virus, which may contribute to genetic stability.
  • conventional seed virus preparations may contain genetically heterogeneous (e.g., mixed) populations of viral sequences, which may over time (e.g., through passage) shift towards certain subpopulation(s) being more dominant. In such an event, the resulting subpopulation that is predominant in the seed virus preparation may differ from the original subpopulation that is predominant in the reference strain.
  • a rescue vaccine of the present invention may be a monovalent influenza vaccine.
  • a regular seasonal vaccine (typically trivalent or quadrivalent) may show that the antigen of one of the strains included in such product is antigenically mismatched.
  • a monovalent rescue vaccine can be produced, which provides a matched antigen, and made available to provide immunoprotection within the same flu season.
  • a regular seasonal vaccine typically trivalent or quadrivalent
  • a rescue vaccine may be produced to include more than one matched antigens accordingly.
  • the rescue vaccine may, for example, be a bivalent rescue vaccine.
  • a rescue vaccine may be produced to include one (or more) matched antigen(s) accordingly.
  • the rescue vaccine may be a monovalent rescue vaccine.
  • a rescue vaccine of the present invention may be un-adjuvanted or adjuvanted.
  • the rescue vaccine may require low dose antigen.
  • Low dose in this context means that the amount of antigen per dose that is required to elicit a statistically acceptable immune response in subjects is less than standard amounts.
  • standard seasonal influenza vaccines contain about 15 ⁇ g of HA per strain in each dosage form.
  • "low dose” vaccines may contain less than about 15 ⁇ g of HA per strain, e.g., about 12 ⁇ g, about 9 ⁇ g, about 7.5 ⁇ g, about 5 ⁇ g, about 3.75 ⁇ g.
  • an adjuvanted rescue vaccine may provide a faster immune response in subjects, as compared to an un-adjuvanted rescue vaccine containing otherwise the same components.
  • a rescue vaccine may still contain a degree of antigenic mismatch (albeit lesser degree than an egg-based vaccines)
  • such rescue vaccine may be adjuvanted, so as to allow broader immune protection in patients despite less-than-perfect antigenic match (e.g., cross protection).
  • an adjuvant may generate a more robust response and increase overall titers to dominant and subdominant epitopes.
  • Adjuvanted rescue vaccine may thus provide a faster immune response in subjects, as compared to an un-adjuvanted rescue vaccine containing otherwise the same components, and may in addition provide antigen sparing effects.
  • the rescue vaccine (or the second influenza vaccine, as defined herein) is adjuvanted, particularly where it contains an antigen with a degree of antigenic mismatch compared to the circulating strain.
  • a degree of mismatch may, for example, be indicated by a 4-fold, 3-fold, 2-fold or 1 -fold difference in HI or a 2-fold or 1 -fold difference in MN titer against the circulating strain compared to the homologous virus titer.
  • a rescue vaccine of the present invention is suitable for immunizing a human subject who has been previously administered a regular seasonal flu vaccine earlier in the flu season.
  • a rescue vaccine of the present invention is suitable for immunizing a human subject who has not been administered with a regular seasonal flu vaccine earlier in the flu season.
  • a rescue vaccine may be used to immunize healthy subjects, subjects at risk, and/or immunocompromised subjects.
  • a rescue vaccine may be used to immunize subjects of various age groups, including the elderly and pediatric populations. Suitable age groups are described further herein.
  • hybrid vaccine refers to a vaccine composition that contains two or more antigens of different production means (e.g., sources or systems).
  • a hybrid vaccine may contain two or more antigens produced from different production platforms.
  • a hybrid vaccine may contain any combinations of: one or more egg-based antigen(s), one or more cell culture-based antigen(s), one or more recombinant protein antigen(s), one or more RNA replicon(s), etc.
  • antigens are provided as a single formulation suitable for administration in human subjects.
  • a full panel of desired antigens may be administered as multiple formulations.
  • at least one of the multiple formulations may be a rescue vaccine (see above).
  • the antigen is an influenza antigen.
  • a hybrid vaccine may be produced to include an antigen prepared in an egg-free process.
  • the egg-free process may be a cell culture, recombinant expression system, an in vitro transcription of RNA replicon, etc. This may be particularly suitable when one or more of the virus strains predicted to be illness-causing viruses in circulation are those known to be susceptible to egg adaptation and/or when a certain clade is known to grow poorly in eggs. This may also be particularly suitable when HA from a certain clade or strain does not bind to red blood cells and/or is not capable of agglutinating red blood cells (e.g. in an HI assay).
  • such virus strain is or comprises H3N2.
  • such virus clade is or comprises Clade 3C.3A or Clade 3C.2A. Further examples of such strains or clades are described herein.
  • an egg-adapted version of a clade results in antigenic mismatch, while a cell version results in greater antigenic match.
  • egg adaptation results in antigenic mismatch through a change in glycosylation pattern on the head region of HA, such as a glycosylation loss, while the glycosylation pattern relevant to antigenicity in cell version is preserved, thus maintaining antigenic match.
  • Relevant glycosylation sites are those contributing to the antigenicity of the antigen, e.g., affecting the presence, accessibility and/or immunodominance of an epitope. For example, Clade 3C.2A may lose key HA sugars upon egg adaptation, which may accompany antigenic mismatch.
  • Antigenic mismatch and greater antigenic match may be determined by HI or MN, as described herein (for example, by >4-fold difference in HI titer against the circulating strain and ⁇ 4-fold difference in HI titer against the circulating strain, respectively, compared to the homologous virus titer).
  • the present invention contemplates that when one or more strains predicted to be disease-causing in an upcoming season are considered to be poor candidates for egg-based production to achieve antigenic match, such strain(s) be produced in egg-free system, such that antigenic match can be achieved in resulting vaccines.
  • one or more antigens produced by egg- free process(es) may be optionally combined with one or more antigens produced in eggs.
  • the invention provides a composition comprising an antigen produced without eggs, which may be an intermediate component to be combined with additional antigen(s) to produce a formulation (e.g., hybrid vaccine) comprising two or more antigens from two or more sources of production methods (e.g., egg- based; cell culture-based, recombinant, RNA replicon, etc.).
  • a formulation e.g., hybrid vaccine
  • sources of production methods e.g., egg- based; cell culture-based, recombinant, RNA replicon, etc.
  • a hybrid vaccine of the invention may comprise two or more components selected from the group consisting of: (i) a viral antigen from an egg-based preparation; (ii) a viral antigen from a cell culture-based preparation; (iii) a viral antigen from a recombinant protein preparation; and (iv) an RNA replicon encoding a viral antigen.
  • the viral antigen may be an influenza antigen.
  • the hybrid vaccine may be trivalent or quadrivalent.
  • the hybrid vaccine may further comprise an adjuvant.
  • the cell culture-based preparation may be a mammalian or avian cell-based preparation. 'Cell culture-based preparation' refers to antigen grown in cell culture.
  • the viral antigen (i) from an egg-based preparation may be a viral antigen which has been passaged in eggs.
  • the viral antigen prepared in an egg-free process may be antigen from a cell culture- based preparation, viral antigen from a recombinant protein preparation and/or viral antigen encoded (in vitro) by a RNA replicon.
  • viral antigen (i) from an egg-based preparation will be from a strain or clade which is not, as described herein, susceptible to egg adaptation and/or to clade mismatch.
  • the viral antigen prepared in an egg-free process ((ii), (iii) and/or (iv)) is antigen from a strain or clade, as described herein, which is susceptible to egg adaptation and/or to clade mismatch.
  • the viral antigen prepared in an egg-free process is antigen from a strain or clade which does not bind to red blood cells and/or is not capable of agglutinating red blood cells (e.g. in an HI assay), as described herein.
  • the viral antigen prepared in an egg-free process may be an influenza antigen (e.g.
  • the reference vaccine may be a first influenza vaccine as defined herein, and/or may have been passaged in eggs.
  • the reference vaccine may, for example, be a vaccine made available earlier in a given influenza season. Relative degree of antigenic match may be determined as described herein (e.g. by HI and/or MN assay).
  • an antigen from an egg-based preparation e.g. an egg-adapted clade or strain
  • an antigen grown in cell culture which, optionally, has not been passaged in eggs
  • the invention aims to overcome existing antigenic mismatch problems by providing vaccination regimes, in which vaccination with a first influenza vaccine comprising an antigen from a first influenza virus, which has been passaged in eggs, is followed by vaccination with a second influenza vaccine comprising antigens from an influenza virus, which has been grown in cell culture.
  • a second vaccine that is more closely antigenically matched to a human circulating influenza strain
  • the methods of the invention provide better protection against influenza, especially in situations where the efficacy of the regular seasonal vaccine is sub-optimal due to the inclusion of a mismatched influenza strain.
  • the invention thus provides a method for immunizing a human, comprising steps of (a) administering to the human a first influenza vaccine comprising an antigen from a first influenza virus which has been passaged in eggs; and subsequently (b) administering to the same human a second influenza vaccine comprising an antigen from a second influenza virus which has been grown in cell culture, wherein the antigen in the second influenza vaccine is more closely antigenically matched to a circulating influenza strain than the antigen in the first influenza vaccine.
  • a method for immunizing a human who has previously been administered a first influenza vaccine comprising an antigen from a first influenza virus which has been passaged in eggs comprising administering to the same human a second influenza vaccine comprising an antigen from a second influenza virus which has been grown in cell culture, wherein the antigen in the second influenza vaccine is more closely antigenically matched to a circulating strain than the antigen in the first influenza vaccine.
  • the antigen from the second influenza virus may not have been passaged in eggs.
  • the invention also provides a first influenza vaccine comprising an antigen from a first influenza virus which has been passaged in eggs and a second influenza vaccine comprising an antigen from a second influenza virus which has been grown in cell culture, for use in immunizing a human by the method of any preceding claim.
  • a first influenza vaccine comprising an antigen from a first influenza virus which has been passaged in eggs for pre-immunizing a human who will receive a second influenza vaccine comprising an antigen from a second influenza virus which has been grown in cell culture, wherein the antigen in the second influenza vaccine is more closely antigenically matched to a circulating strain than the antigen in the first influenza vaccine.
  • a second influenza vaccine comprising an antigen from a second influenza virus which has not been passaged in eggs (e.g. which has been grown in cell culture) for use in immunizing a human who has previously received a first influenza vaccine which has been passaged in eggs, wherein the antigen in the second influenza vaccine is more closely antigenically matched to a circulating strain than the antigen in the first influenza vaccine.
  • the two influenza vaccines are administered within the same influenza season.
  • the second influenza vaccine may be administered 1 month, 2 months, 3 months, 4 months or 5 months after the first influenza vaccine.
  • the second influenza vaccine is administered within 3 months after the first influenza vaccine.
  • the invention also provides a kit comprising: (i) a first influenza vaccine comprising an antigen from a first influenza virus which has been passaged in eggs; and (ii) a second influenza vaccine comprising an antigen from a second influenza virus which was grown in cell culture, wherein the antigen in the second influenza vaccine is more closely antigenically matched to a circulating strain than the antigen in the first influenza vaccine.
  • the kit may comprise instructions for the use of the kit in the methods of the invention.
  • the influenza strain [85] The current process for preparing seasonal vaccines against human influenza virus infection involves the following steps (refs. 1 & 2): (a) isolation of circulating virus strains; (b) antigenic and genetic analysis of isolated viruses; (c) selection of viral strains for use during the coming season; (d) preparation of high-growth seed strains (i.e. CVVs) by reassortment or the use of reverse genetics (or by passage in eggs or cell culture, e.g. for B virus prototype strains that do not require reassortment, as referred to below); (e) release of seed strains (CVVs) to vaccine manufacturers; (f) evaluation by the manufacturers of the strains' suitability for industrial production; and (g) growth of the seed strains (i.e. passage and expansion of the CVVs) to produce virus (i.e. seed virus stocks) from which vaccines are then manufactured.
  • Steps (a) to (e) of this process are performed by the FDA and government-approved international influenza centres, typically under the auspices of the World Health Organization (WHO); steps (f) and (g) are performed by the manufacturers themselves.
  • WHO World Health Organization
  • Steps (d) and (g) transition a virus from one that is naturally adapted for infecting humans into one that will grow to high titers under industrial growth conditions.
  • step (d) typically involves creating a 6:2 reassortant strain that includes the HA and NA encoding genome segments from the strains selected in (c) and the remaining six genome segments from a strain that grows efficiently in chicken eggs, and this strain is usually A/PR/8/34. The reassortment procedure is then followed by repeated passaging of the strain in embryonated eggs to allow for egg adaptation and growth enhancement.
  • prototype strains with good growth characteristics are usually obtained by direct and repeated passaging in embryonated eggs without attempting to generate reassortants.
  • step (d) may instead involve creating a non-6:2 reassortant, for example a 5:3 or a 4:4 reassortant.
  • step (g) the steps performed prior to release to vaccine manufacturers involve passaging influenza virus through eggs. Even if the viruses are grown by a manufacturer in step (g) on a cell substrate, rather than on eggs, the virus will still have been passaged through eggs at some stage between isolation in step (a) and receipt by a manufacturer in step (e).
  • an antigen from an influenza virus which has been grown in cell culture or eggs this generally refers to the growth in step (g).
  • an antigen from an influenza virus which has been grown in eggs or cell culture refers to an antigen that was prepared from an influenza virus that was harvested from eggs or cell culture, respectively.
  • the influenza virus will have been grown in eggs
  • the influenza virus will have been harvested from the eggs and the antigen would have been prepared from the harvested influenza virus.
  • the influenza virus can have been grown in eggs at any stage during the production process, i.e. at any stage during the production process.
  • the step of passaging in eggs is deemed of particular importance by the authorities.
  • the 2003/04 influenza season in the northern hemisphere was dominated by the A/Fujian/41 1/2002-like variant, but the vaccine strain being used contained the H3N2 from the previous year (A/Panama/2007/1999).
  • This strain was a poor antigenic match to the Fujian strain, which led to reduced vaccine effectiveness.
  • the Fujian strain had been rejected by the U.S. Food and Drug Administration (FDA) because it had not been passaged in eggs (refs. 2 & 3), and no antigenically-similar egg-isolated strains were available.
  • FDA U.S. Food and Drug Administration
  • influenza strains found in vaccines need to be matched as closely as possible to the circulating influenza strain.
  • authorities like the WHO and the FDA publish a list of the predominant circulating influenza strains each year and further publish recommended influenza strains for inclusion in influenza vaccines, or reference strains to guide selection of the strains for inclusion in influenza vaccines.
  • influenza antigens used in the first and second influenza vaccines of the invention are matched to a circulating strain. This is necessary in order to allow them to elicit an immune response against said influenza strain.
  • the influenza antigen in the second influenza vaccine will generally provide a closer antigenic match to a circulating influenza strain than the influenza antigen in the first influenza vaccine.
  • Influenza antigens which are antigenically matched generally come from influenza strains which belong to the same influenza subtype (for example H1 N1 , H3N2 or H5N1 ) and are antigenically similar. Whether two influenza antigens are antigenically matched can be easily determined, for example using a hemagglutinin inhibition (HI) assay. In particular, two influenza antigens will be considered antigenically matched if they show a high degree of cross-reaction in a HI assay (e.g., less than fourfold titer).
  • HI hemagglutinin inhibition
  • influenza antigen in the second influenza vaccine is matched more closely to a circulating strain than the influenza antigen in the first influenza vaccine. Again, this can easily be determined by a person skilled in the art through standard assays, like the HI assay. Alternatively, or in addition, the MN assay may be used.
  • non-human animals such as ferrets or mice
  • non-human animals are either infected with a live virus or injected with either the first or the second influenza antigen.
  • blood is extracted from the non-human animals, serum is prepared and subjected to serial dilution (for example a series of 1 :10, 1 :20, 1 :40, 1 :80, 1 :160, 1 :320, 1 :640, 1 :1280, 1 :2560, 1 :5120, 1 :10240 and 1 :20480).
  • Equal volumes of each dilution point are then combined with red blood cells and a circulating influenza strain, wherein the final concentration of red blood cells and the circulating influenza strain is the same for each dilution.
  • the reactions are then carefully monitored for hemagglutination. The higher the dilution at which the antibody can still inhibit hemagglutination the better the antigen can be considered matched to the circulating strain.
  • the highest dilution at which antibodies to the first and the second influenza antigen can still inhibit hemagglutination one can determine which antigen is matched more closely to a circulating strain.
  • Antigenic match may be assessed by infecting the non-human animal with live virus (the first or second virus) which contains the antigen of interest (e.g. the first or second influenza antigen).
  • the live virus may be the seed virus strain from which the first or second influenza vaccine may be manufactured.
  • the antigen in the second influenza vaccine is more closely antigenically matched to a circulating strain than the antigen in the first influenza vaccine if antibodies raised against the antigen in the second influenza vaccine show a higher degree of cross-reaction in an HI assay compared to antibodies raised against the antigen in the first influenza vaccine under identical conditions.
  • the antigen in the second influenza vaccine is more closely antigenically matched to a circulating strain than the antigen in the first influenza vaccine if the highest dilution at which antibodies raised against the antigen in the second influenza vaccine can inhibit hemagglutination is higher compared to the highest dilution at which antibodies raised against the first antigen can inhibit hemagglutination, using an HI assay under identical conditions.
  • an antibody raised against the second influenza antigen can inhibit hemagglutination up to a dilution of 1 :2560 whilst the antibody against the first influenza antigen can inhibit hemagglutination only up to a dilution of 1 :1280, the second influenza antigen will be considered to be matched more closely antigenically. It is understood that "identical” in this context means that all conditions are identical except the antibody which is used in the assay as this antibody will have been raised against the individual antigens.
  • the antigen in the second influenza vaccine is more closely antigenically matched to a circulating strain than the antigen in the first influenza vaccine if the highest dilution at which antibodies raised against the antigen in the second influenza vaccine can inhibit hemagglutination by the circulating strain is closer to the highest dilution at which antibodies raised against the circulating strain can inhibit hemagglutination by the circulating strain (i.e. closer to the homologous virus titer), than the highest dilution at which antibodies raised against the antigen in the first influenza vaccine can inhibit hemagglutination by the circulating strain, under identical conditions.
  • the antigen in the second influenza vaccine may, for example, show ⁇ 4-fold difference in HI titer against the circulating strain compared to the homologous virus titer.
  • the antigen in the second influenza vaccine will show less than 4-fold difference (e.g. ⁇ 3-fold, ⁇ 2-fold, 1 - fold difference) in HI titer against the circulating strain compared to the homologous virus titer.
  • the antigen in the second influenza vaccine will show ⁇ 2-fold difference in HI titer against the circulating strain compared to the homologous virus titer.
  • the antigen in the first influenza vaccine may, for example, show >4-fold difference in HI titer against the circulating strain compared to the homologous virus titer.
  • the antigen in the second influenza vaccine is more closely antigenically matched to a circulating strain than the antigen in the first influenza vaccine if the MN neutralization titer against the circulating strain for antibodies raised against the antigen in the second influenza vaccine is closer to the homologous MN titer than the MN neutralization titer against the circulating strain for antibodies raised against the antigen in the first influenza vaccine, under identical conditions.
  • the antigen in the second influenza vaccine may, for example, show ⁇ 2-fold difference in MN titer against the circulating strain compared to the homologous MN titer.
  • the antigen in the first influenza vaccine may show >2-fold difference in MN titer against the circulating strain compared to the homologous MN titer.
  • an antigen in a second influenza vaccine may show a higher degree of cross-reaction in an HI assay (e.g. a two-way HI test) and/or an MN assay, when compared to a circulating strain, than an antigen in a first influenza vaccine when compared to the circulating strain in said assay(s).
  • the second influenza vaccine may be a rescue vaccine, hybrid vaccine or composition as described herein.
  • the first influenza vaccine may be a seasonal influenza vaccine.
  • the first and second influenza vaccines may comprise antigen from a strain which is susceptible to egg adaptation and/or to clade mismatch.
  • a virus strain or clade that is susceptible to egg adaptation and/or to clade mismatch may be a H3N2 strain.
  • the clade may be Clade 3C.2A (A/Hong Kong/5738/2014-like or A/Hong Kong/4801/2014-like), Clade 3C.3A (e.g. A/Switzerland/9715293/2013-like), or 3C.1 (e.g. A/Texas/50/2012-like virus isolate).
  • the virus strain or clade that is susceptible to egg adaptation and/or to clade mismatch may be a B strain, for example a strain from the B-Victoria lineage (e.g.
  • B/Brisbane/60/2008-like B/Brisbane/60/2008-like.
  • One of ordinary skill in the art can readily identify a strain or clade that is susceptible to egg adaptation and/or to clade mismatch. For example, passaging an isolate of such a strain in eggs may result in virus which is less closely antigenically matched compared to the original isolate and/or circulating strain. In contrast, when the isolate is passaged in mammalian cells, such mismatch in the resulting virus is not observed (or is not observed to the same degree).
  • the circulating strain can be a seasonal circulating strain. Influenza virus strains for use in seasonal vaccines change from season to season. As discussed above, the authorities publish lists of the circulating influenza strains each year and so a skilled person is aware of these.
  • the circulating strain may also be a pandemic influenza strain (i.e. a strain to which the vaccine recipient and the general human population are immunologically naive), such as H2, H5, H7 or H9 subtype strains (in particular of influenza A virus).
  • a pandemic influenza strain i.e. a strain to which the vaccine recipient and the general human population are immunologically naive
  • H2, H5, H7 or H9 subtype strains in particular of influenza A virus.
  • the antigen in the second influenza vaccine is from the same influenza subtype as an antigen in the first influenza vaccine which has been passaged in eggs.
  • the influenza antigen in the second influenza vaccine may be from a H1 strain, a H3 strain or an influenza B strain.
  • the antigen in the second influenza vaccine will also be of the H1 subtype.
  • the antigen in the second influenza vaccine will be of the same subtype as those strains.
  • the antigen in the second influenza vaccine may contain one or both of an influenza antigen from a H1 strain and/or a H3 strain.
  • influenza virus vaccines are generally based either on live virus or on inactivated virus.
  • Inactivated vaccines may be based on whole virions, split virions, or on purified subunit (e.g., purified surface) antigens.
  • Influenza antigens can also be presented in the form of virosomes. The invention can be used with any of these types of vaccine, but will typically be used with inactivated vaccines.
  • the antigen may take the form of a live virus or an inactivated virus.
  • Chemical means for inactivating a virus include treatment with an effective amount of one or more of the following agents: detergents, formaldehyde, formalin, ⁇ propiolactone, or UV light. Additional chemical means for inactivation include treatment with methylene blue, psoralen, carboxyfullerene (C60) or a combination of any thereof. Other methods of viral inactivation are known in the art, such as for example binary ethylamine, acetyl ethyleneimine, or gamma irradiation.
  • the INFLEXALTM product is a whole virion inactivated vaccine.
  • the vaccine may comprise whole virion, split virion, or purified surface antigens (including hemagglutinin and, usually, also including neuraminidase).
  • Virions can be harvested from virus containing fluids by various methods. For example, a purification process may involve zonal centrifugation using a linear sucrose gradient solution that includes detergent to disrupt the virions. Antigens may then be purified, after optional dilution, by diafiltration.
  • Split virions are obtained by treating virions with detergents or solvents (e.g. ethyl ether, polysorbate 80, deoxycholate, tri-/V-butyl phosphate, Triton-X-100, Triton N101 , cetyltrimethylammonium bromide, etc.) to produce subvirion preparations, including the Tween-ether' splitting process.
  • detergents or solvents e.g. ethyl ether, polysorbate 80, deoxycholate, tri-/V-butyl phosphate, Triton-X-100, Triton N101 , cetyltrimethylammonium bromide, etc.
  • Methods of splitting influenza viruses are well known in the art (e.g.. see refs. 4-9, etc.).
  • Splitting of the virus is typically carried out by disrupting or fragmenting whole virus, whether infectious or non-infectious with a disrupting concentration of a splitting agent.
  • Preferred splitting agents are non-ionic and ionic (e.g., cationic) surfactants, e.g., alkylglycosides, alkylthioglycosides, acyl sugars, sulphobetaines, betains, polyoxyethylenealkylethers, ⁇ , ⁇ -dialkyl-Glucamides, Hecameg, alkylphenoxy-polyethoxyethanols, quaternary ammonium compounds, sarcosyl, CTABs (cetyl trimethyl ammonium bromides), tri-/V-butyl phosphate, Cetavlon, myristyltrimethylammonium salts, lipofectin, lipofectamine, and DOT-MA, the octyl- or nonylphenoxy polyoxyethanols (e.g., the Triton surfactants, such as Triton X
  • splitting procedure uses the consecutive effects of sodium deoxycholate and formaldehyde, and splitting can take place during initial virion purification (e.g., in a sucrose density gradient solution).
  • Split virions can usefully be resuspended in sodium phosphate-buffered isotonic sodium chloride solution.
  • the AFLURIATM, BEGRIVACTM, FLUARIXTM, FLUZONETM and FLUSHIELDTM products are split vaccines.
  • Purified surface antigen (or purified subunit) vaccines comprise the influenza surface antigens hemagglutinin and may also include neuraminidase. Processes for preparing these proteins in purified form are well known in the art.
  • the FLUVIRINTM, AGRIPPALTM, FLUCELVAXTM, FLUADTM and INFLUVACTM products are subunit vaccines.
  • strains which can usefully be included in the compositions are strains which are resistant to antiviral therapy (e.g., resistant to oseltamivir (ref. 10) and/or zanamivir), including resistant pandemic strains (ref. 1 1 ).
  • the influenza virus may be attenuated.
  • the influenza virus may be temperature-sensitive.
  • the influenza virus may be cold adapted.
  • the first influenza vaccine can be a trivalent influenza vaccine.
  • the first influenza vaccine can also be a tetravalent influenza vaccine.
  • Such tetravalent influenza vaccines frequently contain two influenza A and two influenza B strains and have the advantage that they can include two different lineages of influenza B viruses. Such vaccines can therefore offer broader protection against circulating influenza viruses.
  • the first influenza vaccine may also include antigen(s) from one, two, five, six or more influenza strains, including influenza A virus and/or influenza B virus. Where a vaccine includes more than one strain of influenza, the different strains are typically grown separately and are mixed after the viruses have been harvested and antigens have been prepared.
  • At least one of the influenza antigens in the first vaccine will have been prepared from an influenza virus that has been passaged in eggs.
  • Human influenza viruses bind to receptor oligosaccharides having a Sia(a2,6)Gal terminal disaccharide (sialic acid linked a 2,6 to galactose), but eggs instead have receptor oligosaccharides with a Sia(a2,3)Gal terminal disaccharide. Growth of human influenza viruses in eggs thus provides selection pressure on hemagglutinin away from Sia(a2,6)Gal binding towards Sia(a2,3)Gal binding.
  • Influenza viruses which have been passaged in eggs can therefore be identified in that they have a binding preference for oligosaccharides with a Sia(a2,3)Gal terminal disaccharide compared to oligosaccharides with a Sia(a2,6)Gal terminal disaccharide.
  • a virus has a binding preference for oligosaccharides with a Sia(a2,3)Gal terminal disaccharide compared to oligosaccharides with a Sia(a2,6)Gal terminal disaccharide
  • various assays can be used.
  • reference 12 describes a solid-phase enzyme-linked assay for influenza virus receptor-binding activity which gives sensitive and quantitative measurements of affinity constants.
  • Reference 13 used a solid phase assay in which binding of viruses to two different sialylglycoproteins was assessed (ovomucoid, with Sia(a2,3)Gal determinants; and pig a2 macroglobulin, which Sia(a2,6)Gal determinants), and also describes an assay in which the binding of virus was assessed against two receptor analogs: free sialic acid (Neu5Ac) and 3' sialyllactose (Neu5Aca2-3Galp1 -4Glc).
  • Reference 14 reports an assay using a glycan array which was able to clearly differentiate receptor preferences for a2,3 or a2,6 linkages.
  • Reference 15 reports an assay based on agglutination of human erythrocytes enzymatically modified to contain either Sia(a2,6)Gal or Sia(a2,3)Gal. Depending on the type of assay, it may be performed directly with the virus itself, or can be performed indirectly with hemagglutinin purified from the virus.
  • influenza vaccine is a multivalent influenza vaccine
  • all of the influenza antigens in the vaccine will usually have been prepared from influenza viruses that have been passaged in eggs.
  • the invention can also be practiced with a first influenza vaccine in which one, two, three, four, five, six or more influenza antigens are prepared from influenza viruses that were passaged in eggs.
  • the first influenza vaccine may also comprise antigens from an influenza virus which has been prepared from influenza viruses grown in eggs. This means that the influenza virus from which the antigen in the vaccine is prepared was grown in eggs.
  • viruses In contrast, where a virus was merely passaged in eggs, it may have been grown in eggs originally and then transferred to cell culture so that the influenza virus from which the influenza antigen is finally prepared was grown in cell culture.
  • Such viruses will retain their binding preference for oligosaccharides with a Sia(a2,3)Gal terminal disaccharide compared to oligosaccharides with a Sia(a2,6)Gal terminal disaccharide and so the methods of the invention will still be beneficial for the efficacy of such vaccines.
  • the antigens in the second influenza vaccine are prepared from an influenza virus grown in cell culture.
  • Growing influenza viruses in cell culture can avoid the selection pressure on hemagglutinin away from Sia(a2,6)Gal binding towards Sia(a2,3)Gal binding which is found when influenza viruses are grown in eggs.
  • it allows for the use of influenza viruses which have not been adapted to high growth in eggs which, again, favors the production of influenza viruses which resemble the circulating strains more closely. Accordingly, these influenza viruses will be a much better match for the circulating human influenza viruses and can thus be expected to provide better protection.
  • the antigen(s) from the second influenza vaccine may be recombinant protein antigen(s).
  • the antigen(s) in the second influenza vaccine has not been passaged in eggs.
  • the second influenza vaccine may be a monovalent influenza vaccine. It may also contain more than one influenza antigen, for example two, three, four, five, six or more influenza antigens provided that at least one influenza antigen was prepared from an influenza virus which has been grown in cell culture. As discussed above, it is understood that the at least one influenza antigen which has been grown in cell culture will be of the same subtype as an influenza antigen in the first influenza vaccine which has been passaged in eggs.
  • the antigen in the second influenza vaccine which has been prepared from an influenza virus grown in cell culture has never been passaged in eggs.
  • antigens can be distinguished from influenza antigens prepared from influenza viruses that have been passaged in eggs by virtue of their preferential binding preference for a Sia(a2,3)Gal terminal disaccharide compared to oligosaccharides with a Sia(a2,6)Gal terminal disaccharide.
  • the viral growth substrate will typically be a cell line of mammalian origin.
  • suitable mammalian cells include, but are not limited to, hamster, cattle, primate (including humans and monkeys) and dog cells.
  • Various cell types may be used, such as kidney cells, fibroblasts, retinal cells, lung cells, etc.
  • suitable hamster cells are the cell lines having the names BHK21 or HKCC.
  • Suitable monkey cells are e.g. African green monkey cells, such as kidney cells as in the Vera cell line.
  • Suitable dog cells are e.g. kidney cells, as in the MDCK cell line.
  • suitable cell lines include, but are not limited to: MDCK; CHO; 293T; BHK; Vero; MRC 5; PER.C6; WI-38; etc.
  • Preferred mammalian cell lines for growing influenza viruses include: MDCK cells (refs. 16-19), derived from Madin Darby canine kidney; Vero cells (refs. 20-22), derived from African green monkey (Cercopithecus aethiops) kidney; or PER.C6 cells (ref. 23), derived from human embryonic retinoblasts.
  • MDCK cells refs. 16-19
  • Vero cells derived from African green monkey (Cercopithecus aethiops) kidney
  • PER.C6 cells derived from human embryonic retinoblasts.
  • PER.C6 is available from the ECACC under deposit number 96022940.
  • Influenza viruses can also be grown on avian cells lines (e.g., refs. 26-28), including avian embryonic stem cells (refs. 26 & 29) and cell lines derived from ducks (e.g., duck retina), or from hens.
  • avian embryonic stem cells include the EBx cell line derived from chicken embryonic stem cells, EB45, EB14, and EB14-074 (ref. 30).
  • Chicken embryo fibroblasts (CEF) can also be used.
  • the most preferred avian cell line is the EB66 cell line, which is derived from duck embryonic stem cells. This cell line has been reported to work well for producing influenza antigens (ref. 31).
  • the cell culture of the present invention is mammalian.
  • Use of mammalian cells is particularly preferable where a strain in the vaccine as described herein is a strain that is susceptible to egg adaptation.
  • a second influenza vaccine e.g., rescue vaccine
  • an egg- free process such as a mammalian cell-based platform.
  • the cells may avoid the selection pressure on hemagglutinin away from Sia(a2,6)Gal binding.
  • certain non-mammalian cells e.g. avian cells
  • non-mammalian cells may be genetically engineered to express a mammalian enzyme or enzymes. Antigen from virus grown in such cells may therefore have a binding preference for oligosaccharides with a Sia(a2,6)Gal terminal disaccharide compared to oligosaccharides with a Sia(a2,3)Gal terminal disaccharide.
  • the most preferred cell lines for growing influenza viruses are MDCK cells.
  • the original MDCK cell line is available from the ATCC as CCL 34, but derivatives of this cell line may also be used.
  • reference 16 discloses a MDCK cell line that was adapted for growth in suspension culture ('MDCK 33016', deposited as DSM ACC 2219).
  • reference 32 discloses a MDCK-derived cell line that grows in suspension in serum free culture ('B-702', deposited as FERM BP-7449).
  • Reference 33 discloses non-tumorigenic MDCK cells, including 'MDCK-S' (ATCC PTA-6500), 'MDCK-SF101 ' (ATCC PTA-6501), 'MDCK-SF102' (ATCC PTA-6502) and 'MDCK-SF103' (PTA-6503).
  • Reference 34 discloses MDCK cell lines with high susceptibility to infection, including 'MDCK.5F1 ' cells (ATCC CRL 12042). Any of these MDCK cell lines can be used.
  • composition will advantageously be free from egg proteins (e.g., ovalbumin and ovomucoid) and from chicken DNA, thereby reducing allergenicity.
  • egg proteins e.g., ovalbumin and ovomucoid
  • the culture for growth, and also the viral inoculum used to start the culture will preferably be free from (i.e., will have been tested for and given a negative result for contamination by) herpes simplex virus, respiratory syncytial virus, parainfluenza virus 3, SARS coronavirus, adenovirus, rhinovirus, reoviruses, polyomaviruses, birnaviruses, circoviruses (in particular porcine circoviruses), and/or parvoviruses (ref. 35). Absence of herpes simplex viruses is particularly preferred.
  • the composition preferably contains less than 10ng (preferably less than 1 ng, and more preferably less than 100 pg) of residual host cell DNA per dose (typically 0.25 ml_ for children or 0.5 ml_ for adults), although trace amounts of host cell DNA may be present.
  • the host cell DNA that it is desirable to exclude from compositions of the invention is DNA that is longer than 100 bp, e.g., DNA fragments longer than 150 bp, longer than 200 bp, etc.
  • the assay used to measure DNA will typically be a validated assay (refs. 36 & 37).
  • the performance characteristics of a validated assay can be described in mathematical and quantifiable terms, and its possible sources of error will have been identified.
  • the assay will generally have been tested for characteristics such as accuracy, precision, specificity.
  • Three principle techniques for DNA quantification can be used: hybridization methods, such as Southern blots or slot blots (ref.
  • ThresholdTM System ref. 39
  • quantitative PCR ref. 40
  • the ThresholdTM system from Molecular Devices is a quantitative assay for picogram levels of total DNA, and has been used for monitoring levels of contaminating DNA in biopharmaceuticals (ref. 38).
  • a typical assay involves non-sequence-specific formation of a reaction complex between a biotinylated ssDNA binding protein, a urease-conjugated anti-ssDNA antibody, and DNA. All assay components are included in the complete Total DNA Assay Kit available from the manufacturer. Various commercial manufacturers offer quantitative PCR assays for detecting residual host cell DNA, e.g., AppTecTM Laboratory Services, BioRelianceTM, Althea Technologies, etc. A comparison of a chemiluminescent hybridization assay and the total DNA ThresholdTM system for measuring host cell DNA contamination of a human viral vaccine can be found in reference 41 .
  • Contaminating DNA can be removed during vaccine preparation using standard purification procedures e.g. chromatography, etc. Removal of residual host cell DNA can be enhanced by nuclease treatment e.g. by using a DNase.
  • a convenient method for reducing host cell DNA contamination is disclosed in references 42 and 43, involving a two-step treatment, first using a DNase (e.g. Benzonase), which may be used during viral growth, and then a cationic detergent (e.g. CTAB), which may be used during virion disruption.
  • Treatment with an alkylating agent, such as ⁇ -propiolactone can also be used to remove host cell DNA, and advantageously may also be used to inactivate virions (ref. 44). Methods using two steps of treatment with an alkylating agent or a combination of a DNase and an alkylating agent have also been described (ref. 45).
  • Vaccines containing ⁇ 10 ng (e.g., ⁇ 1 ng, ⁇ 100 pg) host cell DNA per 15 ⁇ g of hemagglutinin are preferred, as are vaccines containing ⁇ 10 ng (e.g., ⁇ 1 ng, ⁇ 100 pg) host cell DNA per 0.25 ml volume.
  • Vaccines containing ⁇ 10 ng (e.g., ⁇ 1 ng, ⁇ 100 pg) host cell DNA per 50 ⁇ g of hemagglutinin are more preferred, as are vaccines containing ⁇ 10 ng (e.g., ⁇ 1 ng, ⁇ 100 pg) host cell DNA per 0.5 ml volume.
  • the average length of any residual host cell DNA is less than 500 bp, e.g., less than 400 bp, less than 300 bp, less than 200 bp, less than 100 bp, etc.
  • virus may be grown on cells in suspension (refs. 16, 46 & 47) or in adherent culture.
  • a suitable MDCK cell line for suspension culture is MDCK 33016 (deposited as DSM ACC 2219).
  • microcarrier culture can be used.
  • cell culture may be used to isolate and grow virus, from a wild- type sample (e.g. a clinical sample), which virus may be used to produce a vaccine or a composition for use according to the invention.
  • the isolate is preferably an isolate of a circulating strain (obtained from the sample).
  • the isolate may be an isolate of a clade or strain that is susceptible to egg adaptation and/or to clade mismatch. Isolation and growth in cell culture may provide further advantages to the composition and the production process when compared to virus isolated and grown in eggs. Isolation in cell culture may significantly improve isolation rates from clinical samples compared to virus isolated using eggs.
  • Isolation and growth in cell culture may elicit higher growth rates and/or increased virus yield compared to virus isolated and grown in an egg-based process.
  • isolation and growth in cell culture may provide improved genetic stability (as described above), i.e., the ability to retain the genetic sequence of the original clinical samples, without introducing host adaptive mutations that cause antigenic mismatch.
  • the clade or strain is susceptible to egg adaptation and/or to clade mismatch (e.g. a strain from the B/Victoria lineage), and/or where a rescue vaccine (e.g.
  • a second influenza vaccine having a closer antigenic match to a particular circulating strain than a first influenza vaccine is to be provided (particularly when it is to be provided for administration within the same season as the first influenza vaccine).
  • the B strain(s) may be from the B/Victoria lineage and/or the B/Yamagata lineage.
  • the B/Victoria strain is present and is a strain or clade that is susceptible to egg adaptation and/or to clade mismatch.
  • the vaccine may, for example, be a tetravalent vaccine comprising two A strains and two B strains (AABB) or a trivalent vaccine comprising two A strains and one B strain (AAB).
  • the vaccine comprises at least two B strains, e.g. a tetravalent AABB vaccine.
  • Cell culture- based isolation and growth may be particularly advantageous for producing, from clinical samples, multiple B strains for use in producing rescue vaccines and compositions described herein.
  • the B strains produced in cell culture may exhibit isolation rates, growth rates, yield and/or genetic stability that is superior to B strains produced in eggs.
  • the cell culture is preferably MDCK cell culture as described herein (which may be MDCK 33016PF cell culture).
  • Cell lines supporting influenza virus replication are preferably grown in serum free culture media and/or protein free media.
  • a medium is referred to as a serum-free medium in the context of the present invention in which there are no additives from serum of human or animal origin.
  • Protein-free is understood to mean cultures in which multiplication of the cells occurs with exclusion of proteins, growth factors, other protein additives and non-serum proteins, but can optionally include proteins such as trypsin or other proteases that may be necessary for viral growth. The cells growing in such cultures naturally contain proteins themselves.
  • Cell lines supporting influenza virus replication are preferably grown below 37°C (ref. 48) (e.g., 30- 36°C, or at about 30°C, 31 °C, 32°C, 33°C, 34°C, 35°C, 36°C), for example during viral replication.
  • the method for propagating virus in cultured cells generally includes the steps of inoculating the cultured cells with the strain to be cultured, cultivating the infected cells for a desired time period for virus propagation, such as for example as determined by virus titer or antigen expression (e.g., between 24 and 168 hours after inoculation) and collecting the propagated virus.
  • the cultured cells are inoculated with a virus (measured by PFU or TCID50) to cell ratio of 1 :500 to 1 :1 , preferably 1 :100 to 1 :5, more preferably 1 :50 to 1 :10.
  • the virus is added to a suspension of the cells or is applied to a monolayer of the cells, and the virus is absorbed on the cells for at least 60 minutes but usually less than 300 minutes, preferably between 90 and 240 minutes at 25°C to 40°C, preferably 28°C to 37°C.
  • the infected cell culture e.g., monolayers
  • the harvested fluids are then either inactivated or stored frozen.
  • Cultured cells may be infected at a multiplicity of infection ("m.o.i.") of about 0.0001 to 10, preferably 0.002 to 5, more preferably to 0.001 to 2.
  • the cells are infected at an m.o.i of about 0.01 .
  • Infected cells may be harvested 30 to 60 hours post infection.
  • the cells are harvested 34 to 48 hours post infection.
  • the cells are harvested 38 to 40 hours post infection. Nevertheless, determining the optimal harvest time is within the normal capabilities of a person skilled in the art.
  • Proteases typically trypsin
  • the proteases can be added at any suitable stage during the culture.
  • the influenza virus may be a reassortant strain, and may have been obtained by reverse genetics techniques.
  • Reverse genetics techniques e.g., refs. 49-53 allow influenza viruses with desired genome segments to be prepared in vitro using plasmids.
  • it involves expressing (a) DNA molecules that encode desired viral RNA molecules e.g. from poll promoters, and (b) DNA molecules that encode viral proteins e.g. from poll I promoters, such that expression of both types of DNA in a cell leads to assembly of a complete intact infectious virion.
  • the DNA preferably provides all of the viral RNA and proteins, but it is also possible to use a helper virus to provide some of the RNA and proteins.
  • Plasmid-based methods using separate plasmids for producing each viral RNA are preferred (refs. 54-56), and these methods will also involve the use of plasmids to express all or some (e.g., just the PB1 , PB2, PA and NP proteins) of the viral proteins, with 12 plasmids being used in some methods.
  • the use of linear expression constructs is also possible (ref. 57).
  • RNA polymerase I transcription cassettes for viral RNA synthesis
  • a plurality of protein coding regions with RNA polymerase II promoters on another plasmid (e.g., sequences encoding 1 , 2, 3, 4, 5, 6, 7 or all 8 influenza A mRNA transcripts).
  • Preferred aspects of the reference 55 method involve: (a) PB1 , PB2 and PA mRNA encoding regions on a single plasmid; and (b) all 8 vRNA encoding segments on a single plasmid. Including the NA and HA segments on one plasmid and the six other segments on another plasmid can also facilitate matters.
  • poll promoters it is possible to use bacteriophage polymerase promoters (ref. 59). For instance, promoters for the SP6, T3 or T7 polymerases can conveniently be used.
  • bacteriophage polymerase promoters can be more convenient for many cell types (e.g., MDCK), although a cell must also be transfected with a plasmid encoding the exogenous polymerase enzyme.
  • an influenza A virus may include one or more RNA segments from a A/PR/8/34 virus (typically 6 segments from A/PR/8/34, with the HA and NA segments being from a vaccine strain, i.e., a 6:2 reassortant), particularly when viruses are grown in eggs. It may also include one or more RNA segments from a A/WSN/33 virus, or from any other virus strain useful for generating reassortant viruses for vaccine preparation. References 62 and 63 also discuss suitable backbones for reassorting influenza A and B strains.
  • the invention protects against a strain that is capable of human-to-human transmission, and so the strain's genome will usually include at least one RNA segment that originated in a mammalian (e.g., in a human) influenza virus. It may include an NS segment that originated in an avian influenza virus.
  • Hemagglutinin (HA) is the main immunogen in inactivated influenza vaccines, and vaccine doses are standardised by reference to HA levels, typically as measured by a single radial immunodiffusion (SRID) assay.
  • Vaccines typically contain about 15 ⁇ g of HA per strain, although lower doses are also used, e.g., for children, or in pandemic situations.
  • Fractional doses such as 1 ⁇ 2 (i.e., 7.5 ⁇ g HA per strain), 1 ⁇ 4 and 1/8 have been used (refs. 64 & 65), as have higher doses (e.g., 3x or 9x doses (refs. 66 & 67)).
  • vaccines may include between 0.1 and 150 ⁇ g of HA per influenza strain, preferably between 0.1 and 50 ⁇ g, e.g., 0.1 -20 ⁇ g, 0.1 -15 ⁇ g, 0.1 -10 ⁇ g, 0.1 -7.5 ⁇ g, 0.5-5 ⁇ g, etc.
  • Particular doses include, e.g., about 45, about 30, about 15, about 10, about 7.5, about 5, about 3.8, about 1 .9, about 1 .5 ⁇ g, etc. per strain. These lower doses are most useful when an adjuvant is present in the vaccine.
  • the components of the vaccines, kits and processes of the invention e.g., their volumes and concentrations) may be selected to provide these antigen doses in final products.
  • TCID50 median tissue culture infectious dose
  • HA used with the invention may be a natural HA as found in a virus, or may have been modified. For instance, it is known to modify HA to remove determinants (e.g., hyper-basic regions around the cleavage site between HA1 and HA2) that cause a virus to be highly pathogenic in avian species, as these determinants can otherwise prevent a virus from being grown in eggs.
  • determinants e.g., hyper-basic regions around the cleavage site between HA1 and HA2
  • Compositions may include detergent, e.g., a polyoxyethylene sorbitan ester surfactant (known as Tweens'), an octoxynol (such as octoxynol-9 (Triton X-100) or t octylphenoxypolyethoxyethanol), a cetyl trimethyl ammonium bromide ('CTAB'), or sodium deoxycholate, particularly for a split or surface antigen vaccine.
  • the detergent may be present only at trace amounts.
  • the vaccine may include less than 1 mg/ml of each of octoxynol 10, a-tocopheryl hydrogen succinate and polysorbate 80.
  • Other residual components in trace amounts could be antibiotics (e.g., neomycin, kanamycin, polymyxin B).
  • An inactivated but non whole cell vaccine may include matrix protein, in order to benefit from the additional T cell epitopes that are located within this antigen.
  • a non-whole cell vaccine that includes hemagglutinin and neuraminidase may additionally include M1 and/or M2 matrix protein. Where a matrix protein is present, inclusion of detectable levels of M1 matrix protein is preferred. Nucleoprotein may also be present.
  • the antigen in the first influenza vaccine will typically be prepared from influenza virions but, as an alternative, antigens such as hemagglutinin can be expressed in a recombinant host (e.g. , in yeast using a plasmid expression system, or in an insect cell line using a baculovirus vector) and used in purified form (refs. 68 & 69). In general, however, antigens will be from virions.
  • Vaccines used with the invention are pharmaceutically acceptable. They may include components in addition to the antigen and adjuvant, e.g., they will typically include one or more pharmaceutical carrier(s) and/or excipient(s). A thorough discussion of such components is available in reference 70.
  • the carrier(s)/excipient(s) used in mucosal vaccines may be the same as or different from those used in parenteral vaccines.
  • compositions may include preservatives such as thiomersal or 2-phenoxyethanol. It is preferred, however, that the vaccines should be substantially free from (i.e., less than 5 ⁇ g/ml) mercurial material, e.g., thiomersal-free (refs. 8 & 71 ). Vaccines containing no mercury are more preferred.
  • a physiological salt such as a sodium salt.
  • Sodium chloride NaCI
  • Other salts that may be present include potassium chloride, potassium dihydrogen phosphate, disodium phosphate dehydrate, magnesium chloride, calcium chloride, etc.
  • compositions for injection will generally have an osmolality of between 200 mOsm/kg and 400 mOsm/kg, preferably between 240-360 mOsm/kg, and will more preferably fall within the range of 290- 310 mOsm/kg. Osmolality has previously been reported not to have an impact on pain caused by vaccination (ref. 72), but keeping osmolality in this range is nevertheless preferred.
  • Compositions may include one or more buffers.
  • Typical buffers include: a phosphate buffer; a Tris buffer; a borate buffer; a succinate buffer; a histidine buffer; or a citrate buffer. Buffers will typically be included in the 5-20 mM range.
  • the pH of a composition will generally be between 5.0 and 8.1 , and more typically between 6.0 and 8.0, e.g., between 6.5 and 7.5, or between 7.0 and 7.8.
  • a process of the invention may therefore include a step of adjusting the pH of the bulk vaccine prior to packaging.
  • the composition is preferably sterile.
  • the composition is preferably non pyrogenic e.g. containing ⁇ 1 EU (endotoxin unit, a standard measure) per dose, and preferably ⁇ 0.1 EU per dose.
  • the composition is preferably gluten free.
  • the composition may include material for a single immunization, or may include material for multiple immunizations (i.e., a 'multidose' kit). The inclusion of a preservative is preferred in multidose arrangements. As an alternative (or in addition) to including a preservative in multidose compositions, the compositions may be contained in a container having an aseptic adaptor for removal of material.
  • Influenza vaccines are typically administered in a dosage volume of about 0.5 ml, although a half volume (i.e., about 0.25 ml) may be administered to children. For intranasal administration, this total dosage volume can be split between nostrils e.g. 1 ⁇ 2 in each nostril.
  • compositions and kits are preferably stored at between 2°C and 8°C. Typically, they should not be frozen. They should ideally be kept out of direct light.
  • Suitable containers for compositions of the invention include vials, syringes (e.g., disposable syringes), nasal sprays, etc. These containers should be sterile.
  • the vial is preferably made of a glass or plastic material.
  • the vial is preferably sterilized before the composition is added to it.
  • vials are preferably sealed with a latex-free stopper, and the absence of latex in all packaging material is preferred.
  • the vial may include a single dose of vaccine, or it may include more than one dose (a 'multidose' vial), e.g., 10 doses.
  • Preferred vials are made of colorless glass.
  • a vial can have a cap (e.g. , a Luer lock) adapted such that a pre filled syringe can be inserted into the cap, the contents of the syringe can be expelled into the vial (e.g., to reconstitute lyophilized material therein), and the contents of the vial can be removed back into the syringe.
  • a needle can then be attached and the composition can be administered to a patient.
  • the cap is preferably located inside a seal or cover, such that the seal or cover has to be removed before the cap can be accessed.
  • a vial may have a cap that permits aseptic removal of its contents, particularly for multidose vials.
  • the syringe will not normally have a needle attached to it, although a separate needle may be supplied with the syringe for assembly and use.
  • Safety needles are preferred. 1 -inch 23-gauge, 1 -inch 25-gauge and 5/8-inch 25-gauge needles are typical.
  • Syringes may be provided with peel-off labels on which the lot number, influenza season and expiration date of the contents may be printed, to facilitate record keeping.
  • the plunger in the syringe preferably has a stopper to prevent the plunger from being accidentally removed during aspiration.
  • the syringes may have a latex rubber cap and/or plunger.
  • Disposable syringes contain a single dose of vaccine.
  • the syringe will generally have a tip cap to seal the tip prior to attachment of a needle, and the tip cap is preferably made of a butyl rubber. If the syringe and needle are packaged separately then the needle is preferably fitted with a butyl rubber shield.
  • Preferred syringes are those marketed under the trade name "Tip-Lok"TM.
  • Containers may be marked to show a half dose volume, e.g., to facilitate delivery to children. For instance, a syringe containing a 0.5 ml dose may have a mark showing a 0.25 ml volume.
  • a glass container e.g., a syringe or a vial
  • a kit or composition may be packaged (e.g., in the same box) with a leaflet including details of the vaccine e.g. instructions for administration, details of the antigens within the vaccine, etc.
  • the instructions may also contain warnings, e.g., to keep a solution of adrenaline readily available in case of anaphylactic reaction following vaccination, etc.
  • the immune response raised by the methods and uses of the invention will generally include an antibody response, preferably a protective antibody response.
  • Methods for assessing antibody responses, neutralizing capability and protection after influenza virus vaccination are well known in the art. Human studies have shown that antibody titers against hemagglutinin of human influenza virus are correlated with protection (a serum sample hemagglutination-inhibition titer of about 30-40 gives around 50% protection from infection by a homologous virus) (ref. 73).
  • Antibody responses are typically measured by hemagglutination inhibition, by microneutralization, by single radial immunodiffusion (SRID), and/or by single radial hemolysis (SRH). These assay techniques are well known in the art.
  • routes that may be used include, but are not limited to, rectal, oral (e.g., tablet, spray), pharyngeal, buccal, vaginal, topical, transdermal or transcutaneous, intranasal, ocular, pulmonary, etc.
  • oral e.g., tablet, spray
  • pharyngeal buccal
  • vaginal topical
  • transdermal transcutaneous
  • intranasal ocular, pulmonary
  • nasal administration can be, e.g., by spray, drops, aerosol, etc.
  • routes that may be used include, but are not limited to, intramuscular injection, subcutaneous injection, intravenous injection, intraperitoneal injection (where available), intradermal injection, etc., and other systemic routes.
  • the preferred parenteral administration route is by intramuscular injection (e.g., into the arm or leg).
  • the administration regimes according to the invention may be used to treat both children and adults. Influenza vaccines are currently recommended for use in pediatric and adult immunization, from the age of 6 months. Thus the patient may be less than 1 year old, 1 -5 years old, 5-15 years old, 15-55 years old, or at least 55 years old.
  • Preferred patients for receiving the vaccines are the elderly (e.g., >50 years old, >60 years old, preferably >65 years), the young (e.g., ⁇ 5 years old), hospitalized patients, healthcare workers, armed service and military personnel, pregnant women, the chronically ill, immunodeficient patients, patients who have taken an antiviral compound (e.g., an oseltamivir or zanamivir compound; see below) in the 7 days prior to receiving the vaccine, people with egg allergies and people travelling abroad.
  • the vaccines are not suitable solely for these groups, however, and may be used more generally in a population. For pandemic strains, administration to all age groups is preferred.
  • high-risk patients may be selected from patients >65 years of age, ⁇ 5 years old (more preferably ⁇ 2 years old), hospitalized patients, healthcare workers, armed service and military personnel, pregnant women, the chronically ill, immunodeficient patients, and patients who have taken an antiviral compound in the 7 days prior to receiving the vaccine.
  • Vaccines and methods provided herein are useful for providing immune-protection in such high-risk patients.
  • the invention includes methods for immunizing a patient, comprising a step of administering to the patient the composition of any one of the embodiments described herein, wherein the patient is a high-risk patient.
  • compositions of the invention satisfy 1 , 2 or 3 of the European Committee for Medicinal Products for Human Use (CHMP, formerly known as CPMP) criteria for efficacy.
  • CHMP European Committee for Medicinal Products for Human Use
  • these criteria are: (1) >70% seroprotection; (2) >40% seroconversion; and/or (3) a GMT increase of >2.5- fold.
  • these criteria are: (1 ) >60% seroprotection; (2) >30% seroconversion; and/or (3) a GMT increase of >2-fold.
  • CHMP European Committee for Medicinal Products for Human Use
  • the seroconversion rate is the % of subjects who have an HI titre before vaccination of ⁇ 1 :10 and >1 :40 after vaccination. However, if the initial titre is >1 :10 then there needs to be at least a fourfold increase in the amount of antibody after vaccination.
  • compositions of the invention satisfy 1 or 2 of the immunological criteria established for influenza vaccines by the Center for Biologies Evaluation and Research (CBER).
  • CBER Center for Biologies Evaluation and Research
  • these criteria are: (1) the % of subjects achieving an HI antibody titer >1 :40 should be >70%; (2) >40% seroconversion.
  • these criteria are: (1) the % of subjects achieving an HI antibody titer >1 :40 should be >60%; (2) >30% seroconversion.
  • the seroconversion rate is defined as: a) for subjects with a baseline titer >40% seroconversion 1 :10, a 4-fold or greater rise; or b) for subjects with a baseline titer ⁇ 1 :10, a rise to >1 :40.
  • Endpoints may be measured at three weeks (e.g. at day 22) after vaccination (in a two-dose administration schedule, this is typically calculated from after the second dose).
  • the vaccines administered according to the invention are administered during the same influenza season.
  • the influenza season typically lasts from October to May with influenza activity peaks being between December and February.
  • the influenza seasons starts in May, peaks in July and ends in October.
  • the second influenza vaccine may be administered within 1 month, 2 months, 3 months, 4 months or 5 months after the first influenza vaccine.
  • the second influenza vaccine is administered within 3 months after the first influenza vaccine.
  • the vaccines may be administered to patients at substantially the same time as (e.g., during the same medical consultation or visit to a healthcare professional or vaccination center) other vaccines e.g.
  • a measles vaccine at substantially the same time as a measles vaccine, a mumps vaccine, a rubella vaccine, a MMR vaccine, a varicella vaccine, a MMRV vaccine, a diphtheria vaccine, a tetanus vaccine, a pertussis vaccine, a DTP vaccine, a conjugated H. influenzae type b vaccine, an inactivated poliovirus vaccine, a hepatitis B virus vaccine, a meningococcal conjugate vaccine (such as a tetravalent A C W135 Y vaccine), a respiratory syncytial virus vaccine, a pneumococcal conjugate vaccine, etc.
  • Administration at substantially the same time as a pneumococcal vaccine and/or a meningococcal vaccine is particularly useful in elderly patients.
  • vaccines may be administered to patients at substantially the same time as (e.g., during the same medical consultation or visit to a healthcare professional) an antiviral compound, and in particular an antiviral compound active against influenza virus (e.g., oseltamivir and/or zanamivir).
  • an antiviral compound active against influenza virus e.g., oseltamivir and/or zanamivir.
  • neuraminidase inhibitors such as a (3R,4R,5S)-4-acetylamino-5-amino-3(1 - ethylpropoxy)-1 -cyclohexene-1 -carboxylic acid or 5-(acetylamino)-4-[(aminoiminomethyl)-amino]-2,6- anhydro-3,4,5-trideoxy-D-glycero-D-galactonon-2-enonic acid, including esters thereof (e.g. the ethyl esters) and salts thereof (e.g. the phosphate salts).
  • esters thereof e.g. the ethyl esters
  • salts thereof e.g. the phosphate salts
  • a preferred antiviral is (3R,4R,5S)-4-acetylamino-5- amino-3(1 -ethylpropoxy)-1 -cyclohexene-1 -carboxylic acid, ethyl ester, phosphate (1 :1 ), also known as oseltamivir phosphate (TAMIFLUTM).
  • VE vaccine effectiveness
  • CDC Centers for Disease Control and Prevention
  • VE point estimate of 50% means that the flu vaccine reduces a person's risk of developing flu illness that results in a visit to the doctor's office or urgent care provider by 50%. Determining VE is within the normal capabilities of the skilled person.
  • VE may be as determined by a regulatory body such as the US CDC. For example, VE may be determined using the methods described in the prior art (ref.
  • VE (%) is calculated as [1 -adjustedOR]x100, wherein OR is the estimated odds ratio for medically-attended, laboratory confirmed influenza in vaccinated versus non-vaccinated subjects (ref. 1 1 1).
  • the estimated adjustedOR may be determined by logistic regression with adjustment for clinically-relevant confounders.
  • covariates include age, comorbidity, province, week of specimen collection and the interval between influenza-like illness (ILI) onset and specimen collection.
  • the first and/or second influenza vaccine(s) may be un-adjuvanted, or they may be administered with an adjuvant.
  • the adjuvant(s) can function to enhance the immune responses (humoral and/or cellular) elicited in a patient who receives the composition.
  • Some adjuvants are effective for parenteral administration but not for mucosal administration (e.g. aluminum salts), and vice versa, although some adjuvants are effective for both routes. Where adjuvants are used, they will be chosen accordingly.
  • Oil-in-water emulsion adjuvants have been found to be particularly suitable for use in adjuvanting influenza virus vaccines.
  • Various such emulsions are known, and they typically include at least one oil and at least one surfactant, with the oil(s) and surfactant(s) being biodegradable (metabolizable) and biocompatible.
  • the oil droplets in the emulsion are generally less than 5 ⁇ in diameter, and may even have a sub- micron diameter, with these small sizes being achieved with a microfluidiser to provide stable emulsions. Droplets with a size less than 220 nm are preferred as they can be subjected to filter sterilization.
  • the emulsion is uniform.
  • a uniform emulsion is characterized in that a majority of droplets (particles) dispersed therein is within a specified size range (e.g., in diameter). Suitable ranges of specified particle size can be, for example, between 50-220 nm, between 50-180 nm, between 80-180 nm, between 100-175 nm, between 120-185 nm, between 130-190 nm, between 135- 175 nm, between 150-175 nm.
  • the uniform emulsion contains ⁇ 10% of the number of droplets (particles) that are outside of the specified range of diameters.
  • mean particle size of oil droplets in the oil-in-water emulsion preparation is between 135-175 nm, e.g., 155 nm ⁇ 20 nm, as measured by dynamic light scattering, and such a preparation contains not more than 1 x 10 7 large particles per ml_ of the preparation, as measured by optical particle sensing.
  • Large particles as used herein mean those having diameters >1 .2 ⁇ , typically between 1 .2-400 ⁇ .
  • the uniform emulsion contains less than 10%, less than 5%, or less than 3% of the droplets that fall outside of the preferred size range.
  • the mean droplet size of particles in an oil-in-water emulsion preparation is between 125-185 nm, e.g., about 130 nm, about 140 nm, about 150 nm, about 155 nm, about 160 nm, about 170 nm, or about 180 nm, and the oil-in-water emulsion is uniform in that less than 5% of the number of droplets in the preparation fall outside the 125- 185 nm range.
  • the invention can be used with oils such as those from an animal (such as fish) or vegetable source.
  • Sources for vegetable oils include nuts, seeds and grains. Peanut oil, soybean oil, coconut oil, and olive oil, the most commonly available, exemplify the nut oils.
  • Jojoba oil can be used, e.g., obtained from the jojoba bean. Seed oils include safflower oil, cottonseed oil, sunflower seed oil, sesame seed oil and the like. In the grain group, corn oil is the most readily available, but the oil of other cereal grains such as wheat, oats, rye, rice, teff, triticale and the like may also be used.
  • 6-10 carbon fatty acid esters of glycerol and 1 ,2-propanediol may be prepared by hydrolysis, separation and esterification of the appropriate materials starting from the nut and seed oils.
  • Fats and oils from mammalian milk are metabolizable and may therefore be used in the practice of this invention.
  • the procedures for separation, purification, saponification and other means necessary for obtaining pure oils from animal sources are well known in the art.
  • Most fish contain metabolizable oils which may be readily recovered. For example, cod liver oil, shark liver oils, and whale oil such as spermaceti exemplify several of the fish oils which may be used herein.
  • a number of branched chain oils are synthesized biochemically in 5-carbon isoprene units and are generally referred to as terpenoids.
  • Shark liver oil contains a branched, unsaturated terpenoids known as squalene, 2,6,10,15,19,23-hexamethyl- 2,6,10,14,18,22-tetracosahexaene, which is particularly preferred herein.
  • Squalane the saturated analog to squalene
  • Fish oils, including squalene and squalane are readily available from commercial sources or may be obtained by methods known in the art. Other preferred oils are the tocopherols (see below). Mixtures of oils can be used.
  • Surfactants can be classified by their "HLB” (hydrophile/lipophile balance). Preferred surfactants of the invention have a HLB of at least 10, preferably at least 15, and more preferably at least 16.
  • the invention can be used with surfactants including, but not limited to: the polyoxyethylene sorbitan esters surfactants (commonly referred to as the Tweens), especially polysorbate 20 and polysorbate 80; copolymers of ethylene oxide (EO), propylene oxide (PO), and/or butylene oxide (BO), sold under the DOWFAXTM tradename, such as linear EO/PO block copolymers; octoxynols, which can vary in the number of repeating ethoxy (oxy-1 ,2-ethanediyl) groups, with octoxynol-9 (Triton X-100, or t- octylphenoxypolyethoxyethanol) being of particular interest; (octylphenoxy)polyeth
  • Non-ionic surfactants are preferred.
  • Preferred surfactants for including in the emulsion are Tween 80 (polyoxyethylene sorbitan monooleate), Span 85 (sorbitan trioleate), lecithin and Triton X-100.
  • Mixtures of surfactants can be used, e.g., Tween 80/Span 85 mixtures.
  • a combination of a polyoxyethylene sorbitan ester such as polyoxyethylene sorbitan monooleate (Tween 80) and an octoxynol such as t-octylphenoxypolyethoxyethanol (Triton X-100) is also suitable.
  • Another useful combination comprises laureth 9 plus a polyoxyethylene sorbitan ester and/or an octoxynol.
  • Preferred amounts of surfactants are: polyoxyethylene sorbitan esters (such as Tween 80) 0.01 to 1 %, in particular about 0.1 %; octyl- or nonylphenoxy polyoxyethanols (such as Triton X-100, or other detergents in the Triton series) 0.001 to 0.1 %, in particular 0.005 to 0.02%; polyoxyethylene ethers (such as laureth 9) 0.1 to 20 %, preferably 0.1 to 10 % and in particular 0.1 to 1 % or about 0.5%.
  • polyoxyethylene sorbitan esters such as Tween 80
  • octyl- or nonylphenoxy polyoxyethanols such as Triton X-100, or other detergents in the Triton series
  • polyoxyethylene ethers such as laureth 9
  • the most preferred oil-in-water emulsions are squalene-in-water emulsions, preferably submicron squalene-in-water emulsions.
  • Specific oil-in-water emulsion adjuvants useful with the invention include, but are not limited to the following:
  • a submicron emulsion of squalene, polysorbate 80, and sorbitan trioleate The composition of the emulsion by volume can be about 5% squalene, about 0.5% polysorbate 80 and about 0.5% sorbitan trioleate. In weight terms, these ratios become 4.3% squalene, 0.5% polysorbate 80 and 0.48% sorbitan trioleate.
  • the emulsion may include citrate ions, e.g., 10 mM sodium citrate buffer. The citrate ions may be in the aqueous phase. More particularly, the composition of the emulsion by volume can be about 4.6% squalene, about 0.45% polysorbate 80 and about 0.5% sorbitan trioleate.
  • the adjuvant known as "MF59" (refs. 74-76 and 133) is described in more detail in Chapter 10 of reference 133 and chapter 12 of reference 77.
  • Squalene, polysorbate 80 and sorbitan trioleate may be present at a weight ratio of 9750:1 175:1 175.
  • the emulsion may contain 36-42 mg/ml squalene as measured by RP-HPLC; 4.1 -5.3 mg/ml polysorbate 80 as measured by RP-LC; and 4.1 -5.3 mg/ml sorbitan trioleate as measured by RP-LC.
  • Concentrations of about 39 mg/mL squalene, about 4.7 mg/mL polysorbate 80, and about 4.7 mg/mL sorbitan trioleate are typical.
  • mean particle size, as measured by dynamic light scattering, of 155 ⁇ 20 nm is preferred.
  • a Z-average droplet size of between 155-185 nm is preferred, with a polydispersity of ⁇ 0.2.
  • the submicron emulsion comprising squalene, polysorbate 80 and sorbitan trioleate is uniform (see above).
  • An emulsion of squalene, a tocopherol, and Tween 80 may include phosphate buffered saline. It may also include Span 85 (e.g., at 1 %) and/or lecithin. These emulsions may have from 2 to 10% squalene, from 2 to 10% tocopherol and from 0.3 to 3% Tween 80, and the weight ratio of squalene:tocopherol is preferably ⁇ 1 as this provides a more stable emulsion. Squalene and Tween 80 may be present volume ratio of about 5:2.
  • One such emulsion can be made by dissolving Tween 80 in PBS to give a 2% solution, then mixing 90 ml of this solution with a mixture of (5g of DL-a-tocopherol and 5ml squalene), then microfluidizing the mixture.
  • the resulting emulsion may have submicron oil droplets, e.g., with an average diameter of between 100 and 250 nm, preferably about 150-180 nm, e.g., about 150 nm, about 160 nm, about 170 nm or about 180 nm.
  • An emulsion of squalene, a tocopherol, and a Triton detergent may also include a 3d MPL (see below).
  • the Triton detergent of the emulsion can be Triton X-100.
  • the tocopherol can be a-tocopherol.
  • the emulsion may contain a phosphate buffer.
  • An emulsion comprising a polysorbate (e.g. polysorbate 80), a Triton detergent (e.g. Triton X-100) and a tocopherol (e.g. an a-tocopherol succinate):
  • the emulsion may include these three components at a mass ratio of about 75:1 1 :10 (e.g., 750 ⁇ g/ml polysorbate 80, 1 10 ⁇ g/ml Triton X-100 and 100 ⁇ g/ml a- tocopherol succinate), and these concentrations should include any contribution of these components from antigens.
  • the emulsion can comprise polysorbate 80, Triton X-100, and an ⁇ -tocopherol succinate.
  • the emulsion may also include squalene.
  • the emulsion may also include a 3d MPL (see below).
  • the aqueous phase may contain a phosphate buffer.
  • An emulsion of squalane, polysorbate 80 and poloxamer 401 (“PluronicTM L121"): The emulsion can be formulated in phosphate buffered saline, pH 7.4. This emulsion is a useful delivery vehicle for muramyl dipeptides, and has been used with threonyl MDP in the "SAF-1 " adjuvant (ref. 78) (0.05-1 % Thr MDP, 5% squalane, 2.5% Pluronic L121 and 0.2% polysorbate 80). It can also be used without the Thr MDP, as in the "AF" adjuvant (ref. 79) (5% squalane, 1 .25% Pluronic L121 and 0.2% polysorbate 80). Microfluidization is preferred.
  • An emulsion having from 0.5-50% of an oil, 0.1-10% of a phospholipid, and 0.05-5% of a non-ionic surfactant As described in reference 80, preferred phospholipid components are phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, phosphatidic acid, sphingomyelin and cardiolipin. Submicron droplet sizes are advantageous.
  • Additives may be included, such as QuilA saponin, cholesterol, a saponin-lipophile conjugate (such as GPI-0100, described in reference 81 , produced by addition of aliphatic amine to desacylsaponin via the carboxyl group of glucuronic acid), dimethyidioctadecylammonium bromide and/or N,N-dioctadecyl-N,N-bis (2-hydroxyethyl)propanediamine.
  • a non-metabolizable oil such as light mineral oil
  • surfactant such as lecithin, Tween 80 or Span 80
  • Additives may be included, such as QuilA saponin, cholesterol, a saponin-lipophile conjugate (such
  • An emulsion in which a saponin (e.g. QuilA or QS21) and a sterol (e.g. cholesterol) are associated as helical micelles The saponin can be QuilA and/or QS21 , and the sterol can be a cholesterol (ref. 82).
  • An emulsion comprising a mineral oil, a non-ionic hydrophilic ethoxylated fatty alcohol, and a non- ionic lipophilic surfactant:
  • the emulsion may comprise an ethoxylated fatty alcohol and/or polyoxyethylene-polyoxypropylene block copolymer (ref. 83).
  • the emulsions may be mixed with antigen extemporaneously, at the time of delivery.
  • the adjuvant and antigen may be kept separately in a packaged or distributed vaccine, ready for final formulation at the time of use.
  • the antigen will generally be in an aqueous form, such that the vaccine is finally prepared by mixing two liquids.
  • the volume ratio of the two liquids for mixing can vary (e.g., between 5:1 and 1 :5) but is generally about 1 :1 .
  • hemagglutininin antigen will generally remain in aqueous solution but may distribute itself around the oil/water interface. In general, little if any hemagglutinin will enter the oil phase of the emulsion.
  • compositions include a tocopherol
  • any of the ⁇ , ⁇ , ⁇ , ⁇ , ⁇ or ⁇ tocopherols can be used, but a-tocopherols are preferred.
  • the tocopherol can take several forms, e.g., different salts and/or isomers. Salts include organic salts, such as succinate, acetate, nicotinate, etc. D-a-tocopherol and DL- a-tocopherol can both be used.
  • Tocopherols are advantageously included in vaccines for use in elderly patients (e.g., aged 60 years or older, 61 years or older, 65 years or older, etc.) because vitamin E has been reported to have a positive effect on the immune response in this patient group (ref. 84). They also have antioxidant properties that may help to stabilize the emulsions (ref. 85).
  • a preferred a-tocopherol is DL-a-tocopherol, and the preferred salt of this tocopherol is the succinate.
  • the succinate salt has been found to cooperate with TNF-related ligands in vivo.
  • a-tocopherol succinate is known to be compatible with influenza vaccines and to be a useful preservative as an alternative to mercurial compounds (ref. 8). Preservative-free vaccines are particularly preferred.
  • Immunostimulatory oligonucleotides can include nucleotide modifications/analogs such as phosphorothioate modifications and can be double-stranded or (except for RNA) single-stranded.
  • References 86, 87 and 88 disclose possible analog substitutions, e.g., replacement of guanosine with 2'- deoxy-7-deazaguanosine.
  • the adjuvant effect of CpG oligonucleotides is further discussed in references 89-94.
  • a CpG sequence may be directed to TLR9, such as the motif GTCGTT or TTCGTT (ref. 95).
  • the CpG sequence may be specific for inducing a Th1 immune response, such as a CpG-A ODN (oligodeoxynucleotide), or it may be more specific for inducing a B cell response, such a CpG-B ODN.
  • CpG-A and CpG-B ODNs are discussed in references 96-98.
  • the CpG is a CpG-A ODN.
  • the CpG oligonucleotide is constructed so that the 5' end is accessible for receptor recognition.
  • two CpG oligonucleotide sequences may be attached at their 3' ends to form "immunomers". See, for example, references 95 & 99-101 .
  • CpG7909 also known as ProMuneTM (Coley Pharmaceutical Group, Inc.).
  • TpG sequences can be used (ref. 102). These oligonucleotides may be free from unmethylated CpG motifs.
  • the immunostimulatory oligonucleotide may be pyrimidine rich.
  • it may comprise more than one consecutive thymidine nucleotide (e.g., TTTT, as disclosed in ref. 102), and/or it may have a nucleotide composition with >25% thymidine (e.g., >35%, >40%, >50%, >60%, >80%, etc.).
  • it may comprise more than one consecutive cytosine nucleotide (e.g., CCCC, as disclosed in ref.
  • oligonucleotides may be free from unmethylated CpG motifs.
  • Immunostimulatory oligonucleotides will typically comprise at least 20 nucleotides. They may comprise fewer than 100 nucleotides.
  • 3dMPL (also known as 3 de-O-acylated monophosphoryl lipid A or 3-0-desacyl-4'-monophosphoryl lipid A) is an adjuvant in which position 3 of the reducing end glucosamine in monophosphoryl lipid A has been de-acylated.
  • 3dMPL has been prepared from a heptoseless mutant of Salmonella minnesota, and is chemically similar to lipid A but lacks an acid-labile phosphoryl group and a base-labile acyl group. It activates cells of the monocyte/macrophage lineage and stimulates release of several cytokines, including IL-1 , IL-12, TNF-a and GM-CSF (see also ref. 103). Preparation of 3dMPL was originally described in reference 104.
  • 3dMPL can take the form of a mixture of related molecules, varying by their acylation (e.g., having 3, 4, 5 or 6 acyl chains, which may be of different lengths).
  • the two glucosamine (also known as 2- deoxy-2-amino-glucose) monosaccharides are N-acylated at their 2-position carbons (i.e., at positions 2 and 2'), and there is also O-acylation at the 3' position.
  • the group attached to carbon 2 has formula -NH- CO-Chb-CPJR '.
  • the group attached to carbon 2' has formula -NH-CO-CH 2 -CR 2 R 2' .
  • the group attached to carbon 3' has formula -0-CO-CH2-CR 3 R 3' .
  • a representative structure is:
  • Groups R , R 2 and R 3 are each independently -(ChbVChh.
  • the value of n is preferably between 8 and 16, more preferably between 9 and 12, and is most preferably 10.
  • Groups R ' , R 2' and R 3' can each independently be: (a) -H; (b) -OH; or (c) -0-CO-R 4 ,where R 4 is either -H or -(ChbVChh, wherein the value of m is preferably between 8 and 16, and is more preferably 10, 12 or 14. At the 2 position, m is preferably 14. At the 2' position, m is preferably 10. At the 3' position, m is preferably 12.
  • Groups R ' , R 2' and R 3' are thus preferably -O-acyl groups from dodecanoic acid, tetradecanoic acid or hexadecanoic acid.
  • the 3dMPL has only 3 acyl chains (one on each of positions 2, 2' and 3').
  • the 3dMPL can have 4 acyl chains.
  • the 3dMPL can have 5 acyl chains.
  • the 3dMPL can have 6 acyl chains.
  • the 3dMPL adjuvant used according to the invention can be a mixture of these forms, with from 3 to 6 acyl chains, but it is preferred to include 3dMPL with 6 acyl chains in the mixture, and in particular to ensure that the hexaacyl chain form makes up at least 10% by weight of the total 3dMPL, e.g., >20%, >30%, >40%, >50% or more. 3dMPL with 6 acyl chains has been found to be the most adjuvant active form.
  • references to amounts or concentrations of 3dMPL in compositions of the invention refer to the combined 3dMPL species in the mixture.
  • 3dMPL can form micellar aggregates or particles with different sizes, e.g., with a diameter ⁇ 150 nm or >500 nm. Either or both of these can be used with the invention, and the better particles can be selected by routine assay. Smaller particles (e.g., small enough to give a clear aqueous suspension of 3dMPL) are preferred for use according to the invention because of their superior activity (ref. 105). Preferred particles have a mean diameter less than 220 nm, more preferably less than 200 nm or less than 150 nm or less than 120 nm, and can even have a mean diameter less than 100 nm.
  • the mean diameter will not be lower than 50 nm. These particles are small enough to be suitable for filter sterilization. Particle diameter can be assessed by the routine technique of dynamic light scattering, which reveals a mean particle diameter. Where a particle is said to have a diameter of x nm, there will generally be a distribution of particles about this mean, but at least 50% by number (e.g., >60%, >70%, >80%, >90%, or more) of the particles will have a diameter within the range x ⁇ 25%.
  • 3dMPL can advantageously be used in combination with an oil-in-water emulsion. Substantially all of the 3dMPL may be located in the aqueous phase of the emulsion.
  • a typical amount of 3dMPL in a vaccine is 10-100 ⁇ g/dose, e.g., about 25 ⁇ g or about 50 ⁇ g.
  • the 3dMPL can be used on its own, or in combination with one or more further compounds.
  • 3dMPL in combination with the QS21 saponin (ref. 106) (including in an oil-in- water emulsion (ref. 107)), with an immunostimulatory oligonucleotide, with both QS21 and an immunostimulatory oligonucleotide, with aluminum phosphate (ref. 108), with aluminum hydroxide (ref. 109), or with both aluminum phosphate and aluminum hydroxide.
  • composition comprising X
  • a composition comprising X may consist exclusively of X or may include something additional, e.g., X + Y.
  • a process comprising a step of mixing two or more components does not require any specific order of mixing.
  • components can be mixed in any order. Where there are three components then two components can be combined with each other, and then the combination may be combined with the third component, etc.
  • animal (and particularly bovine) materials are used in the culture of cells, they should be obtained from sources that are free from transmissible spongiform encaphalopathies (TSEs), and in particular free from bovine spongiform encephalopathy (BSE). Overall, it is preferred to culture cells in the total absence of animal derived materials.
  • TSEs transmissible spongiform encaphalopathies
  • BSE bovine spongiform encephalopathy
  • a cell substrate is used for reassortment or reverse genetics procedures, it is preferably one that has been approved for use in human vaccine production, e.g., as in Ph Eur general chapter 5.2.3.
  • Influenza viruses include a diverse range of subtypes, classified by surface antigen characteristics. Further variation exists; thus, specific influenza strain isolates are identified by a standard nomenclature specifying virus type, geographical location where first isolated, sequential number of isolation, year of isolation, and HA and NA subtype. Due to a high degree of antigenic variations of surface antigens, it is important that influenza vaccines are prepared to match the dominant circulating (e.g., disease-causing) strains so as to provide sufficient protection.
  • egg-adapted A/Texas/50/2012-like viruses are a weak match for related circulating viruses. It has been recognized that egg-adaptive mutations affect antigenicity. The 2013-14 season recommendation highlighted the importance of ensuring antigenic match and suggested that new systems are needed to enable better match by non-egg-based processes.
  • Figure 1 depicts the relationship between flu-associated illness (as measured by fraction of hospital visits) over the course of a season for each of six influenza seasons. What it indicates is that it is extremely challenging for the system to address antigenic changes. Moreover, egg adaptation may be a driver for unrecognized vaccine antigenic change. It also highlights that by the current system we have in place, effective vaccination against H3N2 has been less than successful. This recognition raises the possibility that in future flu seasons, where H3N2 is predominant, similar antigenic mismatch of available seasonal vaccines may occur. That is, recommendation may be mismatched due to egg adaptation and/or incorrect clade.
  • certain clades are particularly susceptible to causing antigenic mismatch.
  • Clade 3C.3A strain has been chosen for upcoming Northern Hemisphere season. It is noteworthy that egg-adapted version (A/Switzerland/9715293/2013) is mismatched to the cell version - matched 13% of tested viruses as reported by UK WHO Collaboration Center. By comparison, the cell version matched closer to 88% of tested circulating viruses.
  • vaccine efficacy is inversely related to influenza-associated medical needs. This relationship is particularly strong for certain subpopulations of patients, e.g., very young children and the elderly, whose immune system may be premature or suppressed. The same is likely to be said for immunocompromised individuals due to illness or medication.
  • H3N2 viruses have been evolving to not bind red blood cells, as supported in technical difficulties observed in HI assays.
  • a large set of 3C.2A viruses went undetected because of their lack of ability to bind red blood cells (RBCs) , which is essential for Hl-based early screening, and many of the 3C.2A viruses remain uncharacterized because they cannot be tested by the standard HI assay.
  • egg-adapted 3C.2A viruses regain the ability to agglutinate RBCs but lose a key glycosylation and become antigenically mismatched from circulating viruses.
  • Figure 4 shows HA titer of A/Hong Kong/5738/2014 (3C.2A), which was subjected to serial passages in MDCK cells.
  • the result shows that synthetic, mammalian cell-produced viruses did not gain the ability to agglutinate RBCs after passaging, while wild-type mammalian cell-produced viruses acquired the ability to bind RBCs, which coincides with loss of HA glycosylation site by passage 5. All egg-adapted viruses had HA titers.
  • Example 2 Antigenic Characterization of Influenza Viruses Produced Using Synthetic DNA and Novel Backbones.
  • influenza viruses have a high mutation rate in their RNA genomes and exist as complex quasi-species, a property that facilitates their natural drift and continuously challenges vaccine production.
  • Influenza strains that circulate in humans frequently acquire antigenically important mutations to escape immunological pressure, giving rise to new variants that can become dominant and cause seasonal re-infections. These antigenic changes dictate that the influenza vaccine be reviewed annually and updated almost as often.
  • Vaccination is the most effective strategy to protect against seasonal influenza; however, vaccine performance varies from year to year, with decreased effectiveness associated with mismatches between the vaccine antigens and those of circulating strains (refs. 1 10-1 12).
  • mammalian cells particularly the MDCK cell line
  • influenza viruses that can be re-isolated and propagated exclusively in embryonated hen's eggs are recommended as candidates for both mammalian cell-based and egg-based vaccine manufacturing platforms. This standard practice perpetuates the likelihood of producing a vaccine mismatch.
  • influenza viruses propagated in mammalian cells often remain genetically and antigenically similar to the virus present in clinical material (refs. 121 -123).
  • viruses isolated in mammalian cell lines qualified for vaccine production can help maintain antigenic match of the vaccine strain to circulating viruses (ref. 123).
  • MDCK 33016PF cells were maintained as previously described (ref. 124). Wild-type influenza viruses were isolated from clinical samples by World Health Organization (WHO) National Influenza Centers. Egg-based reassortant viruses were generated at WHO Collaborating Centers (WHO CC). All viruses were from stocks held at the Crick Institute, Mill Hill laboratory, UK.
  • WHO World Health Organization
  • WHO CC WHO Collaborating Centers
  • HA and NA segments were assembled as previously described (ref. 124), or with the following modifications.
  • Overlapping oligonucleotides were assembled using primers BMP_13 and BMP_14 (ref. 124). PCR products were denatured and re-annealed to form mismatched duplex DNA, followed by incubation with Surveyor nuclease (Transgenomic, Inc.) and Exonuclease III (NEB).
  • Error-corrected DNA was amplified using nested primers BMP_27 (TTGGGTAACGCCAGGGTTTTCC) (SEQ ID NO: 1) and BMP_34 (TTC AC AC AG G AAAC AG CTATG ACC ATG ATTA) (SEQ ID NO: 2), and purified by ethanol precipitation. Final products are linear gene segments flanked by upstream and downstream regulatory control elements.
  • Synthetic viruses were generated as previously described (ref. 124). Briefly, synthetic HA and NA gene cassettes and plasmids carrying the six backbone genes (PB2, PB1 , PA, NP, M, NS) and the plasmid TMPRSS2 (encoding a serine protease (ref. 125)) were co-transfected into MDCK cells. Clarified culture medium was harvested at least 72 hours post-transfection, and viruses detected by a focus- formation assay (ref. 124). All experiments were performed with viruses rescued and passaged up to 3 times in MDCK cells.
  • HA amino acid sequences used to generate synthetic viruses are listed below in the standard single-letter format:
  • Viral RNA was extracted using the QIAamp Viral RNA Mini Kit (Qiagen) and cDNA generated using Monsterscript reverse transcriptase (Epicentre). HA and NA genes were amplified using Platinum PCR SuperMix High Fidelity DNA polymerase (Life Technologies), and sequences analyzed by Sanger DNA sequencing.
  • Hemagglutination and HI assays were performed according to standard WHO methods by using 0.1 % suspensions of guinea pig red blood cells and 20 nM oseltamivir carboxylate. HA titers were determined in the presence of drug. HI titers were reciprocals of the highest dilutions of sera that inhibited hemagglutination. Post-infection ferret antisera against various reference viruses were treated with receptor-destroying enzyme from Vibrio cholera.
  • PR8x Three optimized backbones (PR8x, #19, and #21) derived from low pathogenicity viruses (ref. 124) were used to make subtype A viruses.
  • the PR8x backbone contains six internal genome segments from an MDCK-adapted A/Puerto Rico/8/1934 strain.
  • the #19 backbone contains PB2, PB1 , and NP from an MDCK-adapted A/Hessen/105/2007 strain and the remaining segments from PR8x.
  • the #21 backbone contains an A/California/07/2009 PB1 and the remaining segments from PR8x.
  • Subtype B viruses were made using all six backbone segments from B/Brisbane/60/2008. Rescued viruses were passaged up to three times in MDCK cells exclusively. The HA and NA genomes of all viruses were confirmed to have 100% genetic identity to the coding sequences used for synthesis (sequences provided above). Viruses generated for antigenicity testing covered seasonal influenza strains (A/H1 N1 , A/H3N2, and B-Victoria lineage), including four egg- and mammalian cell-derived matched pairs ( Figure 5). The H3N2 subtype was prioritized in this study given that H3N2 candidate vaccine viruses (CCVs) used in vaccine production have failed to match circulating strains for the past several years and continue to present a challenge.
  • CCVs H3N2 candidate vaccine viruses
  • antisera raised against the CWs recognized the corresponding synthetic viruses at titers ⁇ 2-fold different from the homologous virus titers, regardless of the backbone used. All the synthetic viruses also reacted similarly in HI assays with ferret antiserum raised against the egg-adapted wild-type strains, with titers ⁇ 4-fold different from the homologous virus titers.
  • antigenic characterization was performed by HI tests using ferret antisera raised against the cell- or egg-propagated wild-type strains. Viruses made with the different backbones reacted similarly to a given antiserum, and HI titers obtained from all the tested viruses were within ⁇ 4-fold of the homologous virus titer.
  • Synthetic viruses can improve match to strains that cause human disease
  • Synthetic viruses can be made using HA and NA sequences from either egg- or mammalian cell- grown isolates, but the ability to match a mammalian cell-grown virus genetically can be expected to improve antigenic match to strains circulating in the human population. It has been documented that egg- selected changes in the HA gene for the recent H3N2 and B-Victoria lineage CWs used in seasonal vaccines have been associated with reduced vaccine effectiveness (refs. 1 10 & 1 19). Therefore, we used synthetic technology to assess differences in antigenicity between mammalian cell- and egg-derived matched antigens for three recent H3N2 strains and one B-Victoria lineage strain.
  • A/Victoria/210/2009 was the H3N2 component for the 2010-1 1 and 201 1 -12 vaccines; A/Victoria/361 /201 1 -like was the H3N2 component for the 2012-13 and 2013-14 vaccines; and A/Switzerland/9715293/2013 was the H3N2 recommendation for the 2015-16 vaccine.
  • B/Brisbane/60/2008 was the B strain component for the 2009-10, 2010-1 1 , and 201 1 -12 trivalent vaccines, and has been the B- Victoria component for quadrivalent vaccines since 2012-13.
  • the HA sequences of all mammalian cell- and egg-derived antigen pairs differed by 1 -3 amino acids (see Table 2 below).
  • Table 2 Sequence differences in synthetic egg- and mammalian cell-derived antigen-matched pairs. position cell egg
  • all the egg-derived H3 antigens contained a G186V mutation that improves virus growth in eggs but alters antigenicity relative to an MDCK cell-propagated virus (ref. 126).
  • the egg-derived B/Brisbane/60/2008 HA loses a potential glycosylation site near the receptor binding site. The loss of this glycan upon egg adaptation affects antigenicity (ref. 1 19).
  • Synthetic viruses made from either mammalian cell- or egg-derived HA and NA sequences were compared by HI assay to their matched synthetic counterpart and to conventional reference strains using antisera raised against mammalian cell- and egg-grown reference viruses. As shown in Figures 8 and 9, in most instances we observed differences in reactivity between the cell- and egg-derived antigens when analyzed with ferret antisera raised against the egg-grown viruses, but not when analyzed with antisera raised against the mammalian cell-grown viruses.
  • ferret antisera raised against the egg- grown vaccine viruses generally recognized synthetic test viruses containing the egg-adapted HA sequence at a titer within 2-fold of the homologous titers, but reacted less well in HI assays (>4-fold decrease) to all viruses expressing the corresponding mammalian cell-derived antigens.
  • This experimental design models the current human immunization situation, in which an immune response against an egg-derived vaccine antigen is intended to protect against circulating strains that lack egg adaptations.
  • ferret antisera raised against the cell-grown isolates recognized viruses expressing either the cell- or egg-derived antigens.
  • Ferret antisera raised against viruses with one backbone also effectively recognized viruses rescued on a different backbone, confirming that the use of alternative backbones with the same HA and NA sequence does not alter antigenicity.
  • the cell-derived synthetic and wild-type antigens reacted better to antiserum raised to the synthetic virus expressing egg- adapted HA (RG-PS-2404) than to antiserum raised to the egg-adapted reference virus ( ⁇ 2-fold versus 4 to 8-fold lower than the homologous titers, respectively).
  • the drifted H3N2 strain A/Texas/50/2012 vaccine strain was antigenically distinct from dominant circulating strains (refs. 1 12 & 120). Furthermore, variability of H3N2 influenza virus isolation rates in eggs in recent years (refs. 127 & 128) can add additional risk of mismatch to the current system. Therefore, the use of CWs isolated or synthetically generated in certified mammalian cells could help increase the number of viruses available for vaccine virus selection and, in some circumstances, provide better matched viruses for vaccine manufacture.
  • MDCK cells have both a-2, 6- and a-2, 3-linked sialic acids on their surfaces, making them a more neutral substrate with respect to selection of altered variants of influenza virus (ref. 1 17). Sequence analysis of influenza viruses in clinical samples and their laboratory-passaged derivatives have confirmed that MDCK cells are more likely than chicken eggs to maintain the prevalent HA genotypes present in clinical material (ref. 122). MDCK cell-grown viruses are also more antigenically similar to the viruses replicating in humans than their egg-grown counterparts, as evidenced by their greater recognition by neutralizing and HI antibodies in post-infection human sera (refs. 129 & 130).
  • H3N2 viruses which may have HA molecules with low affinity for cell surface sialic acid
  • some H3N2 viruses have been reported to acquire upon passage a mutation in the NA sialic acid binding site that facilitates binding to MDCK cells (ref. 132).
  • the selected NA mutation does not interfere with the antigenic and immunogenic properties of the mutated viruses, per se, the altered properties of NA result in NA- mediated hemagglutination and consequent changes to HI titers.
  • the NA inhibitor oseltamivir was added to the HI assays for all H3N2 strains in the studies we are reporting.
  • Example 3 Exploring serological cross-reactivity of mouse antisera induced by vaccination with cell-derived and egg-adapted monovalent H3N2 vaccines.
  • the designation “egg” or “cell” refers to the passage history of the viruses that provided the HA and NA sequences for synthesis. In cases of mixed passage history, any passage in eggs is sufficient to trigger an "egg" designation.
  • HA levels were quantified by SRID and HA sequences were confirmed. SRID values are based on A/Switzerland9715293/2013 reagents.
  • Vaccines prepared from the monobulks were administered to BALB/c mice (10 mice per group), as shown in Table 4 (with or without MF59 adjuvant).
  • HI and MN assays were used to study serological cross-reactivity between the mouse antisera and the viral antigens. MN results are shown in Figures 1 1 A and 1 1 B. The data shown were generated using the day 41 bleed (3 weeks post second immunization). In general, antibody titers were highest against the homologous virus. MF59 increases overall antibody titers (in both HI and MN assays), but specificity is unchanged. Notably, there was a clear egg-cell antigenic mismatch for the clade 3C.2a A/Hong Kong/5738/2014 (A/HK) vaccine (e.g. compare 3rd and 4th columns of Figure 1 1A with 1 1 B).
  • test virus when the test virus was A/HK containing the egg-adapted HA and NA sequences, the virus was neutralized by all tested mouse antisera (Figure 1 1 A), including antisera to both the homologous egg-derived A/HK virus antigen and the cell-derived A/HK virus antigen.
  • Figure 1 1 B when the test virus was A/HK containing the cell-derived HA and NA sequences, the virus was neutralized by the homologous cell-derived A/HK antisera, but not by the antisera to the egg-derived A/HK virus antigen.
  • Example 4 Animal study to investigate monovalent, inactivated influenza A virus (H3N2) vaccine as a rescue vaccine.
  • H3N2 monovalent, inactivated influenza A virus
  • H3N2 cell-derived monovalent, inactivated influenza A virus
  • TIV Egg-derived trivalent
  • MIV cell-derived monovalent vaccines were administered to BALB/c mice (10 mice per group), as shown in Table 5 (with or without MF59 adjuvant).
  • TIV contains egg-derived A/Switzerland/9715293/2013 (H3N2), A/California/07/2009 (H1 N1) and B/Brisbane/9/2014 virus antigens.
  • MIV contains cell-derived A/Hong Kong/5738/2014 (H3N2) virus antigen.
  • the TIV antigens were from egg-derived monobulks equivalent to those used to manufacture a trivalent influenza vaccine administered to humans during the 2015-2016 (northern hemisphere) seasonal vaccination campaign.
  • the MIV antigens were produced in cell culture from a synthetic seed virus using HA and NA sequences from a cell-derived A/HK virus (i.e. a virus which has not been passaged in eggs).
  • mice antisera e.g. obtained from the day 41 and/or day 63 bleeds
  • viral antigens in a similar manner to Example 3, using HI and MN assays.
  • MN and HI titers are to be obtained using the antisera from each group, tested against the cell-derived A/HK virus (from which the MIV antigen is derived) and the A/Switzerland egg-derived virus (from which the H3N2 component of the TIV is derived).
  • the study is expected to provide confirmation that administration of the better matched cell-derived MIV as a rescue vaccine will advantageously improve neutralizing antibody titers in vivo against the corresponding circulating strain, compared to TIV administration alone.

Abstract

L'invention concerne des compositions vaccinales contre la grippe, des préparations associées et des intermédiaires, des formulations, des procédés de production, des procédés d'immunisation, et leur utilisation, pour obtenir une protection immunitaire chez des sujets humains. Plus spécifiquement, l'invention concerne des vaccins contre la grippe non à base d'oeufs, qui permettent une correspondance antigénique améliorée.
EP16738554.1A 2015-06-26 2016-06-24 Vaccins contre la grippe à correspondance antigénique Withdrawn EP3313439A2 (fr)

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